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Analysis of functional domains of the packaging proteins of bacteriophage T3 by site-directed mutagenesis
J. Moi. Biol. (1994) 235,248-259
Analysis of Functional Domains of the Packaging Proteins of Bacteriophage T3 by Site-directed Mutagenesis Miyo Morita, Masao Tasaka and Hisao Fujisawat Department of Botany, Faculty of Science Kyoto University, Kyoto 606, Japan
Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gpl8 and gpl9. We have developed a defined system, composed of purified gpl8, gpl9 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate, gpl9 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by sitedirected mutagenesis, gpl9 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging ativity in the defined and crude systems: group 1 mutants were defective in both systems (gpl9-G61D, which is a gpl9 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group4 mutants had almost the same activity as gpl9-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective" interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.
Keywords: T3 phage; DNA packaging protein gpl9; site-directed mutagenesis; functional domains
1. I n t r o d u c t i o n
jointed together in a head-to-tail fashion through the terminally redundant sequences. During packaging of DNA, mature monomers are cut from the concatemer. DNA packaging is thought to proceed by a common mechanism for most dsDNA phages (Murialdo & Becker, 1978; Earnshaw & Casjens, 1980). Formation of precursors for DNA translocation, prohead/packaging enzyme/DNA complexes, depends upon either ATP as an allosteric effector (T3; Shibata el al., 1987a) or ATP hydrolysis (~t; Becket et al., 1977; 429; Guo et al., 1987a). DNA translocation into the prohead is driven by ATP hydrolysis (Black, 1989). Packaging
During head assembly of most double-stranded (ds:~) DNA bacteriophages, DNA is packaged into the cavity of a preformed protein shell, called the prohead, with the aid of non-capsid protein, called a terminase or packaging enzyme. DNA is synthesized as concatemers, in which unit-length molecules are t Author to whom all correspondence and reprint requests should be addressed. :~ Abbreviations used: ds, double-stranded; gp, gene product; wt, wild-type; peq, phage equivalents. 0022-2836/94/010248-12 $08.00/0
248 © 1994 AcademicPress Limited
Mutant D N A Packaging Proteins of Phage T3 enzymes (terminases) and in vitro packaging systems display a DNA dependent ATPase activity. These results indicate that ATP plays multiple roles in the DNA packaging reaction. In general, the packaging enzyme is commonly composed of two subunits; the small subunit has DNA binding activity and the large subunit has prohead binding activity. It is thought that the large subunit carries the crucial functions, whereas the small subunit is stimulative for packaging of mature DNA (Black, 1989). In general, the large subunits have the ATP binding and potential Mg2+ binding motifs (Guo et al., 1987b). In the case of 2, the small subunit also has ATP binding motifs (Becket & Gold, 1988) and displays a DNA independent ATPase (Parris et al., 1988). To elucidate the DNA packaging mechanisms at the molecular level, we have constructed a defined and a crude in vitro system for packaging phage T3 DNA. The crude system contains extracts of T3-infected cells and DNA is packaged by way of concatemer as an intermediate (Fujisawa et al., 1978). The defined system is composed of mature T3 DNA, purified proheads and two packaging proreins, gpl8 and gpl9, corresponding to the small and large subunits of the packaging enzyme, respectively (Hamada et al., 1986b). Our previous results indicate the sequential events in DNA packaging: the packaging proteins gpl8 and gpl9 bind DNA and proheads, respectively (Shibata et al., 1987a). These complexes associate to form a direct precursor complex (50 S complex) for DNA translocation into the head. The formation of the gpl9-prohead complex and the 50S complex requires ATP as an allosteric effector. The DNA translocation into the head is driven by hydrolysis of ATP. The defined in vitro DNA packaging system displays an ATPase which is composed of DNA packaging:dependent and -independent ATPases (pac- and nonpac-ATPases, respectively: Morita et al., 1993).
gpl9 is only an ATP-binding protein in the defined packaging system (Hamada et al., 1987) and has putative ATP and Mg2+-binding consensus sequences (Fig. 1 and Table 1; see Guo et al., 1987b). Therefore, it is expected that gpl9 is involved in ATP interaction in formation of the gpl9-prohead and the 50 S complexes, and in ATP hydrolysis in DNA translocation. Previously, we have undertaken a molecular genetic approach to dissect functional domains of gpl9 by site-directed mutagenesis into these ATP and metal binding motifs, and mutant proteins were partially characterized. Two important mutants were isolated: mutant gpl9 with a Gly to Asp mutation at amino acid 61 (gpl9-G61D) was defective in DNA packaging, probably due to an altered interaction with ATP; and gp19-G424E, with a mutation in another putative ATP binding domain, was active in DNA packaging but defective in cutting of the concatemer (Kimura & Fujisawa, 1991). These results show that the ability to inactive function(s) of gpl9 by single amino acid substitution is useful in studies to understand the mechanisms of the DNA packaging in which gpl9 plays a central role. In this paper, we have expanded the previous studies on gpl9 by more extensive analysis by sitedirected mutagenesis to define the role(s) of large subunit gpl9 in the packaging machinery. The results suggest that the ATP binding domain-I (Gly61) and Mg2+ binding domain-I (His344) are involved in the gpl9-ATP interaction, and that the ATP-binding domain-III (Gly429) is important in coupling of ATP hydrolysis to DNA translocation gpl9 is responsibile for processing of concatemers in concert with multiple ATP-binding domains. 2. M a t e r i a l s a n d M e t h o d s
(a) Bacteria, phages and plasmids Escherichia coli CJ236 (dut 1 ung 1 thi 1 ,'el AI; pCJl05 (Cmr)) was used for preparation of M13 viral DNA
Table 1 Comparison of A T P binding sequences of the T3 D N A packaging protein gp19 and other proteins
Proteins
Residues
T3 gpl9
52-72 360-372 484-505 154-174 293-314 164-184 60-80
2gpA T4 gpl7 DnaA RecA ATPase = of E. coil Myosin of rabbit
164-184
173-192
T3 gpl9 Bovin PKA RSV p60"re
418-435 44-62 269-287
EGF receptor FSV ==='rp~
690-707 923-941
249
Amino acid sequences KKF-ILQAFRGIGKSFITCAFVV ILVIDPSGRGKDE RVI-PIKGASVYGKPVASMPRKR _RMTVCNL_S-RQLGKTT-VVAIFL HFY-DIWTAAVEGKSGFEPYTAI Y_NPLFLYGGTGLGKTT-FAMNLV _R-IVEIYG PGSSGKTT-LTLQVI _R--ELIIGDRGTGKTALAIDAII _S--ILITGESGAGKTV-NTKKVI VFESNFGDGMFGKVFSPVL ERIKTLGTGSFGRVMLVKH RLEAKLG_G_Q_GCFGEVWMGTW KI-KVLGSGAFGTVYKGLW LLGERIGRGNFGEVFSGRL
Residues showing substantial homology between gpl9 and other ATP binding proteins are underlined.
M u t a n t D N A Packaging Proteins of Phage T 3
250 0
containing uracil (Kunkel et al., 1987). E. coli JMl03 was used as a host (Ung+~ for most of the p~asmid constructs derived from MI3 m p l 9 (Yanish-Perron et al., 1985). A host for expression vector pNT45, which uses ). PL promoter for expression of cloned genes, is described in a previous paper (Hamada et al., 1986a). Amber mutants of genes 3 (endonuclease, 3-), 5 (DNA polymerase, 5-) and 19 (a large subunit of packaging proteins, 19-) of T3 phage were from our laboratory stocks, p K H 2 is a pNT45 derivative carrying a 2"32 kb fragment containing gene 19, which is an expression vector for gene 19 (Hamada et al.~ 1986a). pUC18 E1-TR is pUCl8 carrying an insert of about 500 bp t h a t includes the terminally redundant sequence and its flanking sequences from T3 eoneatemeric DNA (TR sequence), plus 3-1 kb including gene 19 (El sequence: Hashimoto & Fujisawa, 1988). Plasmid was iinearized by PstI digestion. (b) Buffers Buffer PM is l0 mM potassium phosphate (pH 7-4) with 7 mM 2-mercaptoethanol; buffer PMG is a buffer PM with 5 % (v/v) glycerol. Complete pac buffer is 5 % (w/v) polyethylene glycol (M, 6000), 100/~M ATP, 20 mM Tris" HCI (pH 7"4), 50 mM NaCl, 1 mM spermidine, 5 mM MgC! 2, 7 mM 2-mercaptoethanol. Prohead buffer is described in a previous paper (Nakasu et al., 1983).
an Applied Biosystems 3804B-02 DNA synthesizer. Each oligonucleotide carried l or 2 mismatched nucleotides that created both an amino acid substitution and a new restriction site at the target region {Fig. l(B)). The termini of oligonuc[eotides were phosphorylated by T4 polynucleotide kinase. M13 m p l 9 viral DNA that carried gene 19 was propagated in E. coil CJ236. To this viral DNA, containing uracil, oligonucleotides were hybridized. and extended with the use of T4 DNA polymerase. T4 DNA ligase was used to close the stands, and the products were introduced into E. coli JMl03 cells. The resulting plaques were isolated and mutants were screened by analysis of newly generated restriction sites. The mutation sites were confirmed by DNA sequencing. (f) In vitro D N A packaging reaction in the defined system A standard reaction mixture (20pl) contained l0 I° phage equivalents (peq) of mature T3 DNA or 0"5 pg DNA, 4 x l0 l° peq of proheads, 20pmol of g p l 8 and 3 pmol of g p l 9 in complete pac buffer (Shibata et al., 1987). After the reaction, the mixture was treated with DNase I (0-2 mg/ml) for 20 min on ice to digest unpackaged DNA, if necessary, and mature heads were converted to infectious particles by incubation with a head acceptor extract containing tail and tail fiber proteins, and phages were assayed by titration on E. coli BB as described (Shibata et al., 1987). Alternatively, DNA in the head was subjected to agarose gel electrophoresis as described in a previous paper (Shibata et al., 1987).
(c) Purification of phage proteins involved in
D N A packaging Proheads and g p l 8 were purified as described in a previous paper (Hamada et al., 1980a). (d) Purification of 9p19 g p l 9 s were purified according to Kimura & Fujisawa (1991). However, some m u t a n t gpl9s (gp19-K64T and gplg-H344D), which were incorporated into inclusion bodies, were purified as following. Cells from 2 1 of overexpressed culture were suspended in 20 m] of PM buffer containing 2 mM EDTA and frozen at - 8 0 ° C . Frozen cells were thawed at 30°C, mixed with 2 ml of lysozyme (10 mg/ml) and kept on ice for 1 h. After addition of 2-5 ml of 5 M NaCI and 0-005 ml of l M MgCI 2, the lysate was sonieated and centrifuged at 8000revs/min in a sorval SS-34 rotor for 15 min. The pellet was suspended in 15 ml" buffer PMG, sonicated and centrifuged as above. The pellet was suspended in 2 ml buffer PMG and combined with 18 ml of 7 M guanidine" hydrochloride in 20 mM phosphate buffer (pH 7"4), l mM DTT. After 30 min on ice, the mixture was dialyzed against I I of 0-2 M NaC1, l mM MgCI 2 in buffer PMG for 6 h and then against I I of 0"1 M NaCl, 1 mM MgC12 in buffer PMG overnight. The lysate was centrifuged to remove aggregates. The supernatant was loaded onto a DEAE-eellulose column (DE52, 5 ml), equilibrated with 0"l M NaCI in buffer PMG, and eluted by a gradient of 0"l M to 0"3 M NaCI in buffer PMG. The procedure hereafter was the same as t h a t for purification of wild-type g p l 9 (Kimura & Fujisawa, 1991).
(e) Oligonucleotide-directed mutagenesis Site-directed mutagenesis was carried out according to the protocol of Kunkel et al. (1987) (Kimura & Fujisawa, 1991}. Primers (33 to 36 bases long) were synthesized by
(g) In vitro D N A packaging in the crude system The reaction mixture contained g p l 9 and 19- extract, a mixture of 3 - / 1 9 - and 5 - / 1 9 - extracts prepared as described elsewhere (Fujisawa & Yamagishi. 1981). supplemented with 2 mM ATP. After the addition of T3 wild-type DNA, the mixture was incubated for 60 min at 30°C. The reaction was stopped by adding chloroform, and after appropriate dilution, phages were titrated on E. coli ER22 (Fujisawa et al., 1978). (h) Isolation of gp19-prohead complexes g p l 9 and proheads were incubated in complete pac buffer with or without ATP at 30°C for 30 min at a molar ratio of g p l 9 to a prohead of 40. After incubation, the reaction mixture was sedimented through 2 ml of 10% (w/v) sucrose in pac buffer with or without ATP in an Hitachi RPS50 at 26,000 revs/min for 90 rain. Pellets were dissolved in sample buffer and subjected to SDS/polyacrylamide gel electrophoresis (Laemmli, 1970). (i) Assay of ATPa~es For ATPase assay, the same standard reaction mixture for in vitro DNA packaging in the defined system as described above was used. ATPase assays were performed as described in a previous paper (Morita et al., 1993). Briefly, the reaction mixture containing [y-32P]ATP at 1-5MBq/ml (100/~M) was incubated with or without actinomycin D (l juM) for 30min at 30°C. Two pl of the reaction mixture was spotted onto PEI-cellulose (Maehery-Nagel. Co.) and dried. The chromatogram was soaked in methanol for 5 min, redried and run in I M formic acid, 0"5 M LiCI. The distribution of radioactivity on ATP and Pi was determined by an image analyzer (Ambis Image Analyzer). The pac- and nonpac-ATPase
Mutant DNA Packaging Proteins of Phage T3
A)
251
100
20O
3O0
400
500
I
I
I
I
I
ml i
I
t
I ATP binding I HLQAFR~IQ._KSF G61D G63D K64T
Mg -binding I _HSYHSC H344D H347R
T
c
586
m I
ATP binding HI ATP binding II ILVIDPSGR.Q_KDE NF~MFfi.K~ G424E G367D G429R G369D G429L K370I K430T
TI Mg -binding II
HM~tP[H I-I553L
B) Restriction enzyme 369 370 367 wild type 5'- CCT AGT GGT CGA GGT AAG GAT -3' P S G R G K D G367D
~ ~ 3 A S D R
G369D G
R
D
Nru l
Figure 1. Site-directed mutagenesis of gpl9. (A) The relative positions of ATP binding and Mg2+ binding motifs and mutated amino acids, which are underlined. Amino acid residues were numbered from the intiation Met (1) predicted from DNA sequences (Yamada et al., 1986). (B) Examples of amino acid substitution and new restriction enzyme sites (boxed) created by nucleotide substitutions (arrow heads).
activities were a difference between total and actinomycin D-resistant ATPases and the actinomycin D-resistant ATPase. respectively. 3. Results
(a) Construction of amino acid substitution mutants Starting from pKH2, the 2-32 kb region, carrying the gene 19, was inserted into the BamHI-EcoRI region in the multicioning site of M13 mpl9. To the resulting construct, the synthetic oligonucleotides were hybridized, and olgonucleotide-directed routsgenesis was carried out by the method of Kunkel et al. (1987). The sequences of the oligonucleotides were designed not only to replace a single amino acid but also to create a new restriction site in the ATP binding and Mg2+ binding domains (Fig. I(B)), as described in a previous paper (Kimura & Fujisawa, 1991). This made it easy to screen for the amino acid substitution mutants, and nine mutants were newly isolated in the present paper. Hereinafter, gpl9 of the wild-type and its derivatives carrying mutations described above are abbreviated as gpl9-wt and gp19-G63D, etc.
after induction by temperature shift-up, proteins expressed from the cloned genes were identified through SDS/polyacrylamide gel electrophoresis. In addition to the previous four mutants (gpl9-G61D, gp19-G424E, gp19-K430T and gp19-H553L: Kimura & Fujisawa, 1991), nine mutants (gplgG63D, gp19-K64T, gp19-H344D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-K370I, gp19G429L and gp19-G429R) were purified to near homogeneity by the standard method (Kimura & Fujisawa, 1991). However, two mutant proteins (gp19-K64T and gp19-H344D) were incorporated into inclusion bodies when overexpressed. These mutant proteins were solubilized by guanidinehydrochloride from inclusion bodies and purified by the standard procedure as described in Materials and Methods. gpl9-wt was also incorporated into including bodies by a longer incubation at 42°C, gpl9-wt purified from inclusion bodies had the same specific activities of DNA packaging in the defined and crude systems as gpl9-wt purified by the standard method (data not shown).
(c) D N A packaging activity (b) Overexpression and purification of 9ene product First, the 2"32 kb fragment was generated from the M13 clones by Sau3A-EcoRI digestion and inserted into the BamHI and EcoRI sites of the expression vector pNT45 in the proper orientation (Hamada et al., 1986a). The resulting constructs were introduced into ER22 (pNT203) cells, and
To assay DNA packaging activity, gpl9-wt or mutant was added to a crude or a defined in vitro system for DNA packaging and assayed by measuring plaque-forming units as described in Materials and Methods. As shown in Table 2, gpl9-G61D, gp19-G63D, gp19-H344D, gp19-H347R, gp19-G367D, gpl9-
Mutant DNA Packaging Proteins of Phage T3
252 Table 2
D N A packaging in the defined ,and crude in vitro systems Packaging activity (% of WT) gpl9
Crude system
Defined system
0' 16 100 0"054 0"47 96"7 3'9 l'21 0"47 1"72 65"8 2"6 35"5 27"3 69'6 126
0"00 100 0"4 25"l 104 l "7 55"2 23"2 77'0 96"1 121"6 98"3 16'3 65"9 110-5
-WT G61D G63D K64T H344D H347R G367D G369D K370I G424E G429L G429R K430T H553L
(e) Specific cleavage activity
DNA packaging reaction was performed by using the defined and crude in ~itrosystems as described in Materials and Metbods. G369D and gp19-G424E were defective in DNA packaging in the crude in vitro system. In the defined system, gpl9-G61D and gp19-H344D were defective, gp19-G63D, gp19G367D and gp19-G429R had decreased activity, and the other m u t a n t s had almost the same activity as gpl9-wt. Accordingly, gp19-G63D, gp19-H347R, gp19-G367D and gp19-G369D were defective in DNA packaging in the crude system but active in the defined system as previously observed with gp19-G424E ( K i m u r a & Fujisawa, 1991). (d) Non-specific endonuclease activity g p l 9 - w t has a non-specific endonuclease activity, suppressible by either A T P or gpi 8 (Fujisawa et al., 1990). To examine the endonuclease activity of m u t a n t gpl9s, circular pUCI8 E I - T R was incu-
~
ATP
+ ' -
+
-
+
bated with or without ATP. Mutants defective in the crude system but. active in the defined system (gp19-G63D, gp19-367D, gpi9-G369D), except gp19-H347R, lacked the endonuclease activity (Fig. 2). Other m u t a n t s showed almost the same activity as gpl9-wt, except t h a t the endonuclease activity of gpl 9-H344D was not suppressed by A T P (data not shown) as is t h a t of gplg-G61D ( K i m u r a & Fujisawa, 1991).
In the defined in vitro I)NA packaging system. linearized plasmid DNAs carrying the concatemer junction are specifically cleaved at the left end of the terminally r e d u n d a n t sequence when packaged leftward, indicating t h a t the cleavage reaction corresponds to the termination cleavage of concatemer processing (Fujisawa et al., 1990). As shown in Figure 3, the products in the cleavage reaction with g p l 9 - w t were a fragment of 3"0 kb, containing the terminally r e d u n d a n t sequence and the remainder of the plasmid, a 2"5 kb fragment, and both fragments were protected from DNase I digestion as shown in a previous paper (Kimura & Fujisawa, 1991). The same results were obtained with gp19-K3701, gp19-G429R, gp19-G429L and gp19-K430T. When the reaction was carried out with m u t a n t s defective in the nonspecific endonuclease activity (gp19-G367 D, gp19-G369D and gp19-G424E), the cleavage was not detected, but when the reaction mixtures were treated with DNase I to digest unpackaged I)NA the 3-0kb fi'agment containing the terminally r e d u n d a n t sequence appeared, d e m o n s t r a t i n g that DNA was packaged leftward and stopped at the left end of the terminally r e d u n d a n t sequence without being cleaved. With gp19-H344D, the cleavage was not. detected and all DNA was digested by DNase I (data not shown), as observed with gpl9-G611) (Kimura & Fujisawa, 1991), d e m o n s t r a t i n g t h a t DNA was not packaged.
r ~.
-
+
-
~
ATP
+
~
-
+
~
-
+
-
+
i
linear
--
lmem"
Figure 2. Endonuelease activity ofgpl9. Closed circular pUCI8 EI-TR DNA w a s incubated with gpl9 in the presence (+) or absence ( - ) of ATP in pac buffer at 30°C for 60 min and subjected to agarose gel electrophoresis. The lmsition of closed circular tee), nicked circular (nc) and linear forms of pUC EI-TR are indicated.
Mutant DNA Packaging Proteins of Phage T3 A
-DNaseI
+DNasel
-DNaseI
253 +DNaseI
N
,
~
~
4730bp! 2920bp 2580bp 2080bp
Cleavage
B
Psi11
W 2.5kb
~l~ I~
3.0kb
Pstl I
~[
direction of packaging Figure 3. Cleavage of the coneatemer joint in the defined in vitro DNA packaging system. (A) Linearized pUCI8 EI-TR was ineubated in the standard reaction mixture containing 250/~M ATP at 30°C for 60 min and sample buffer or 1)Nase I was added. Samples with DNase I were incubated on ice for 20 rain before addition of sample buffer. DNAs were then subjected to agarose gel eleetrophoresis as described in Materials and Methods. Sizes of the molecular weight standards are indicated. (B) A diagram of the packaging and cleavage reaction. The direction of DNA packaging and a site of I)NA cleavage are indicated by arrows.
(f) ('haracterization of gp19 mutants in the D N A packaging reaction in the defined in vitro system |)uring DNA packaging in the defined system, gpl9 and gpl8 form separate complexes with proheads and DNA, respectively. These two complexes form a 50 S complex and then DNA translocation is driven by hydrolysis of ATP. The formation of the g p l 9 - p r o h e a d and the 50S complex depends upon ATP as an allosteric effector (Shibata et al., 1987a). To characterize the defect in DNA packaging of the group 1 mutants, the g p l 9 - p r o h e a d interaction was analyzed by determining copy numbers of gpl9 in g p l 9 - p r o h e a d complexes, g p l 9 - p r o h e a d complexes were isolated and subjected to SDS/ polyacrylamide gel electrophoresis as described in Materials and Methods. Relative band densities of gpl9 to gpl8 of Coomassie brilliant blue-stained gel were calibrated by a densitometer to estimate copy numbers in the complexes as described in a previous paper (Fujisawa et al., 1991). As shown in Figure 4, we confirmed the previous results that gpl9-wt bound to a prohead at molar ratios of 6 or 20 in the presence or absence of ATP, respectively (Fujisawa et al., 1991). However, gpl9-G61D bound to a prohead at a molar ratio of 20, independent of the presence or absence of ATP. gpl 9-H344D bound to a prohead at ratios of more than 6 to 20, indepen-
dent of the presence or absence of ATP (data not shown). These results indicate that group 1 mutants are abnormal in prohead binding activity. To characterize gpl9 mutants, the effect of gpl9 mutation on the rate of DNA translocation was examined. The defined standard reactions were incubated in the presence of ATP-y-S at 30°C for five minutes to allow formation of the precursor 50 S complex; DNA packaging was initiated at 20°C by diluting the reaction mixture 100 times into complete pac buffer containing ATP. The reaction was terminated by the addition of DNase I and mature heads were assayed as described in Materials and Methods. With all mutants, the kinetics of the appearance of mature heads was not different from that of gpl9-wt, indicating that the DNA translocation rate was not affected by gpl9 mutants (data not shown). However, with gp19-G429R and gpl9-G429L (Fig. 5), the conversion of most 50 S complexes into mature heads took a much longer time, and with gp19-G429R mature head production decreased two orders of maginitude when started from the 50 S complex.
(g) Tryptic digestion of mutant gp19 The cleavage patterns of gpl9-wt by several proteases were altered by the addition of ATP,
254
Mutant DN.4 Packaging Proteins of Phage T3
f~
!
WT II +
+ATP
G61D
I -
I
+
•
WT
ATP p
', K64T
' G61D ' G63D
I 2 3 4 511 2 3 4 5 1 1 2 3 4 511 2 3 4 5
gpl6 gpl5 - - gpl9
67kl)
' - x ~, 8
45kD 39kD
gp9, gpl0
'i
gpl0
E Trypsin
(a)
-- ATP u
WT
~, G61D
i 2 3 4 5',1 2 3
~:'~::-~-~-~ ~ : ~=*~*"< ., "
'~ G63D ~ K64T
4 5',12 3 4
s',~ 2 3
u
4 5 r-
' *~i:'~:. *;~~~'~ --67kD
Figure 4. SDS/polyaerylamide gel electrophoresis of gpl9-prohead complexes. Mixtures containing proheads and 40 molecules per prohead of gpl9-wt or gpl9-G61D in pac buffer were incubated in the presence ( + ) or absence ( - ) of ATP at 30°C for 30 min. gplg-prohead complexes were prepared as described in Materials and Methods and subjected to SDS/polyacrylamide gel electrophoresis.
--45kD --39kD
i (b)
0 8
H344D
WT I
I
I I -ATP
+ATP
-ATP
M I 2 3 4 5 2 3 4 5 I 23
÷ATP
4 5 2 345
M 97.4kl)
~"
6-
66.2k1)
45kl)
5"
31kD
0
10
20
Time (rain) Figure 5. Time-course of formation of mature heads from the 50 S complex. A standard reaction containing ATP-7-S in place of ATP was ineubat~d at 30°C for 5 min to allow formation of the 50 S precursor complex and then diluted I00 times into the complete pac buffer containing ATP at 20°C to initiate DNA transloeation into the head. The reaction was stopped by the addition of DNase I at the indicated times (min) and mature beads were assayed as described in Materials and Methods. Wild-type (O); G429L (0); G429R ( , ) . P.F.U., plaque-forming units.
21.5kl)
(c)
Figure 6. Effect of ATP on the cleavage of gpl9 by trypsin, gpl9 (2-5 g/20/~l) was exposed in the presence or absence of ATP (50 FM) to 2/~g/20 Fl or 0"l Fg/20 Fl of trypsin, respectively, for various times at 20°C (lanes l, 0 rain; 2, 5 min; 3, l0 rain; 4, 30 rain; 5, 90 rain). Portions were subjected SDS/polyacrylamide gel electrophoresis.
Mutant DNA Packaging Proteins of Phage T3 indicating that ATP induces a conformational change in gpl9 (Fujisawa et al., 1991). The functional change(s) induced by a mutation might be related to a change(s) in the conformation of gpl9, reflecting an altered sensitivity to proteases. To examine the protease sensitivity of gpl9 mutants, gpl9s were incubated in the presence or absence of ATP at 20°C and subjected to limited digestion with trypsin at 20°C. As shown in Figure6(A) and (B), the digestion pattern of gpl9-wt by trypsin was affected by the addition of ATP as described in a previous paper (Fujisawa et al., 1991). gpl9-G61D and gp19-H344D (Fig. 6(A), (B). and (C)) were more sensitive to trypsin than gpl9-wt. Their digestion patterns were the same as each other and different from that of gpl9-wt but not significantly affected by the addition of ATP. gp19-G63D, gp19-K64T (Fig. 6(A) and (B)) and other gpl9 mutants (data not shown) were indistinguishable from gpl9-wt in their sensitivity to trypsin, the digestion patterns and the effect of ATP on the digestion patterns. (h) pac-ATPase activity As shown in a previous paper (Hamada et al., 1987), the defined in vitro DNA packaging system displays a DNA-dependent ATPase activity. But the ATPase activity is stimulated by non-packageable DNA such as single-stranded or circular DNA or even RNA (nonpac-ATPase). Among inhibitors of DNA packaging, actinomycinD specifically inhibited the ATPase, strictly coupled to DNA translocation into the prohead (pac-ATPase). The
Table 3 pac- and nonpac-ATPase activities of the defined in vitro system 32p-ATP hydrolyzed(nmol/ml)
gp|9
~n-p~-ATPase
pac-ATPase
WT ATP-binding I G61D G63D K64T Mg"+-binding I H344D H347R ATP-binding II G367D G369D K370I ATP-binding III G424E
27"8+4"2
17'6+ 1-6
19.0+3.1 N.D. N.D.
1'6+0-6 N.D. N.D.
24"0+ 3"8 N.D.
2-04-0"5 N.D.
24-2+4"6 22"0+3"5 68"0+10-1
13"0+2"1 13"2-t-1"1 17"0+1"9
25"6+ 5'2
G429L G429R K430T Mg2 +-binding
24"2 4- 4"2 27"4 4- 5"0 42"2 4- 6"2
16"64-1"8 25"4 4- 2"3 24-2 + 3"3 17"6_+_1"8
N.D.
N.D.
H533L
II
The pac- and nonpac-ATPase activities of the defined in vitro system were measuredas described in Materialsand Methods.
255
pac-ATPase activity is, therefore, defined as the difference between total and actinomycin D-resistant, nonpac-ATPases (Morita et al., 1993). To examine the effect of gpl9 mutations on pacand nonpac-ATPase, the ATPase activities were determined in the presence or absence of actinomycin D. As shown in Table 3, gplg-G61D and gp19-H344D, defective in DNA packaging in the defined system, lacked pac-ATPase activity, although the nonpac-ATPase activity was not affected, gp19-G429R had an increased pac-ATPase activity, in spite of the decrease in the DNA packaging activity. There was no mutant defective in the nonpac-ATPase, although gp19-K370I and gp19430T had an increased activity of the nonpacATPase.
4. D i s c u s s i o n
As observed among several dsDNA phages, the packaging enzyme is commonly composed of two subunits. The small is implicated in DNA binding and stimulates DNA packaging. The large subunit binds to the prohead and appears to be crucial for DNA packaging (Black, 1989). In general, the large subunit has consensus sequences for ATP binding and potential Mg 2+ binding regions (Guo et al., 1987b). Analysis of DNA packaging in the defined system of T3 phage indicates that ATP has multiple functions in DNA packaging as an allosteric effector in formation of two precursor complexes, the gpl9-prohead complex and the 50 S (prohead/packaging enzyme/DNA) complex, and as an energy source in DNA translocation into the head (Shibata et al., 1987a,b). In ). (Becker et al., 1977) and ~b-29 (Guo et al., 1987a), the intermediates in the DNA packaging process are unstable in the presence of non-hydrolyzable ATP analogs and are stabilized by ATP hydrolysis. A necessary approach to understanding the molecular mechanism of DNA translocation driven by ATP hydrolysis is the characterization of interaction between ATP and the packaging machinery, gpl9, the large subunit of the T3 packaging enzyme, is only an ATP binding protein in the packaging system (Hamada et al., 1987) and ATP induces a conformational change in gpl9 (Fujisawa et al., 1991). gpl9 carries ATP and Mg2+ binding motifs. A series of mutations were introduced into both ATP and Mg2+ binding motifs of gpl9 and analyzed. Table 4 summarizes properties of gpl9 mutants, including the previously isolated four mutants (Kimura & Fujisawa, 1991). gpl9 mutants were divided into four groups according to the DNA packaging activity in the defined and crude systems (Tables 2 and 4): group 1 mutants were defective in both systems (gplg-G61D, gplg-H344D); a group 2 mutant had decreased activity in both systems (gplg-G429R); group 3 mutants were defective only in the crude system (gp19-G63D, gplg-H347R, gpl9-G3fi7D, gp19-G369D, gp19-G424E); group4 mutants had almost the same activity as gpl9-wt
Mutant DNA Packaging Proteins of Phage T3
256
Table
4
Summary of the properties of mutant gp19s Packaging activity
Functions
lmc-ATPase •Group
Crude
Defined
activity
Interaction with ATP
WT G61D
++
++
++
+
H344D
--
,G429R
+
gpl9
--
DNA cleavage
Prohead binding
Motif
+
+
+ +
+ +f + + f
M-i
+
A-i
+
+ + +
+
N.I).
A-Ill
+
N.D.
+
N.I).
--
+
+
+
N.D.
G424E H347R
--
+ + + + + +
+ + + N.D.
+ + +
+
N.I). N.I). N.I).
A-I A-I I A-If
K64T K370I G429L K430T H553L
++ ++ + ++ ++
N.D. ++ +++ ++ N.I).
+ + + + +
+ + + + +
N.I). N.I). N.i). N.I). N.I).
G63D G367D G369D
+ + + + +
+ + + + +
A-III
M-I A-I A-If A-Ill A-Ill M-II
Data are summarized by qualitatively representing mutant activities relative to gpl9-wt activities as + + (DNA packaging and pewATPase) or + (others): packaging activity. Table 2: pac-ATPase activity. Table 3: interacti¢m with ATP. Figs 2. 6 and unpublished results: DNA cleavage, Figs 2, 3 and unpublished results: prohead binding. Fig. 6. and unpublished results. ATP and Mg2÷-binding motifs are abbreviated as A and M, respectively. N.D. not determined. t Inaet.ive binding.
(gp19-K64T, gp19-K370I, gp19-G429L, gp19K430T, gp19-H553L). Digestion p a t t e r n s of g p l 9 by several proteases were altered by the addition of ATP, indicating t h a t g p l 9 changes its conformation upon binding to A T P (Fujisawa et at., 1991). With the group 1 m u t a n t s , their trypsin digestion p a t t e r n s were different from t h a t of g p l 9 - w t in the absence of A T P and not significantly altered by the addition of A T P (Fig. 6). With gpl9-G61D, a b o u t 20 molecules bound to a prohead, independent of the presence or absence of ATP, unlike g p l 9 - w t for which six molecules bind the prohead in the presence of A T P and 20 bind in the absence of A T P (Fig. 4). The non-specific endonucleolytic activity of g p l 9 - G 6 1 D was suppressed by gpl8, like t h a t o f g p l 9 - w t , but not suppressed by A T P . u n l i k e t h a t of g p l 9 - w t ( K i m u r a & Fujisawa. 1991), indicating t h a t the gpl9--ATP interaction is lost in gpl9-G61D, gp19-G63D, another m u t a n t in the A T P binding domain-I, had decreased DNA packaging activity in the defined in vitro system. Gly61 and Gly63 are located in the A T P binding domain t h a t is conserved a m o n g the large subunits of packaging proteins of m a n y phages (Guo et al., 1987b; Table 1). These facts suggest t h a t A T P binding d o m a i n - I is involved in ATP-binding. With gp19-H344D, a b o u t 20 molecules bind to a prohead, independent of the presence or absence of ATP, but the ratios varied with experiments, suggesting t h a t the H344D m u t a t i o n m a y induce a conformational change in g p l 9 , resulting in unstable binding to a prohead. His 344 constitutes a potential Mg 2+ binding domain, His-X2-His (Berg, 1986; Guo et al., 1987b), which is conserved between T3 and T7 (Dunn & Studier, 1983; Y a m a d a et al., 1986). The
requirement of Mg 2+ for T3 DNA packaging has been d e m o n s t r a t e d (Fujisawa et al., 1978). Tile complete defect in DNA packaging activity of gpl 9H344D suggests t h a t this domain is involved in interaction with Mg 2+. g p l 9 - G 6 1 l ) - p r o h e a d or g p i 9 - H 3 4 4 D - p r o h e a d complexes were defective in DNA packaging. I t is reasonable t h a t these m u t a n t s lost the pac-ATPase activity because I)NA packaging is not initiated with these mutants. From the observations t h a t the tryptic digestion patterns of these m u t a n t s were different from t h a t of g p l 9 - w t , the possibility t h a t an amino acid sul)stitution causes a conformational change in the g p l 9 molecules, resulting in impaired activity, not by disruption of the predicted activity, A T P or Mg 2+ binding activity, cannot be excluded. The same a r g u m e n t would hold true for following discussions. Only one group 2 m u t a n t , gp19-G429R, had an increased pac-ATPase activity in spite of one-fifth i)NA packaging activity of g p l 9 - w t in both tile crude and defined systems ( T a b l e s 2 and 3). Therefore, gp19-G429R would require eight times more A T P for translocation of the same length of DNA than gpl9-wt. I t should be noted t h a t gp19-G429L, another substitution m u t a n t of Gly429, had an increased pac-ATPase activity (Table 3). These results suggest t h a t Gly429 in the A T P binding d o m a i n - I l l is i m p o r t a n t in coupling of A T P hydrolysis to I)NA translocation. With these mutants, although a lag period before the first appearance of m a t u r e heads was not different fi'om t h a t of gp 19-wt, it took a longer time tbr the conversion of most 5 0 S complexes to m a t u r e heads (Fig. 5), indicating t h a t the rate of DNA translocation slows down, on average, or t h a t it takes a
Mutant DNA
P a c k a g i n g Proteins of Phage T 3
much longer time for initiation of DNA translocation, starting from the precursor 50 S complexes. With gp19-G429R, the phage yields decreased more severely when started from the 50 S complex (Table 2 and Fig. 5), suggesting that the 50S complex containing gp19-G429R may be unstable and be inactivated during incubation with ATP-~-S. The group 3 mutants were active in DNA packaging in the defined system, but defective in the crude system. In the crude system, DNA is packaged by way of concatemer as an intermediate and monomeric I)NA must be cut from the concatemer (Fujisawa et al., 1978). The group 3 mutants except gp19-H347R were defective in both the non-specific endonuclease activity and the specific cleavage activity which is coupled to DNA packaging as gp19-G424E (Kimura & Fujisawa, 1991). The fact that these defects were produced by single amino acid substitutions indicate that the same domains of gpl 9 are responsible for the specific and non-specific cleavage activities. The group 3 mutations except the H347R mutation would lead to the defect in the con(.atemer processing, without affecting their ability to package mature T3 DNA. The mechanism which changes the non-specific cleavage activity to the specific cleavage activity necessary for" processing of concatemers remains to be elucidated. There are at least three activities required for the specific cleavage: cleavage sequence recognition, nucleotide binding and phosphodiester bond cleavage. Although cleavage of the concatemer junction was not observed, the longer fragment containing the terminally redundant sequence appeared when the reactions were treated with 1)Nasel (Fig. 3), indicating that the cleavage sequence, the left end of the terminally redundant sequence, is recognized and bound specifically, but not cleaved by these mutants. Since DNA cleavage activity is controlled through interaction with ATP, mutations in the ATP binding domains may induce a conformational change of gpl9 to inactivate the cleavage activity. Mutations in group3, except H347R, are scattered among the three ATP binding domains. These results suggest that these domains are close to each other in the gpl9 molecule to constitute a single functional domain or that several domains are involved in the endonucleolytic activity. The location of the endonuclease activity domains in the large subunit of the packaging proteins may be general (T4, Rao & Black, 1988: 2, l)avidson & Gold, 1992). In ),, mutations in two distinct domains of the large subunit gpA inactivate the endonuclease activity of the terminase, without disrupting the DNA packaging activity (Davidson & Gold. 1992). In addition to gpA, the cleavage at cos sequences depends on the small subunit gpNul, which is involved in recognition and binding of the co.~ sequence (Frackman et al., 1985). Mutations in or near the ATP binding motif of gpNul alter the interaction of terminase with the cos sequence (Feiss et al., 1988; Cue & Feiss, 1992). In spite of being a group 3 mutant, gpl9-H347R
257
was active in both endonuclease activities. Since the specific endonuclease activity in the defined system corresponds to the termination cleavage of the concatemer processing (Fujisawa et al., 1990), this mutant would be defective in the initiation events. We have not characterized the group 4 mutants ill detail because these mutants were active in DNA packaging in both the defined and crude systems. Although the importance of Lys to ATP binding domains is clear (Fry et al., 1986; Maruyama & MacLennan, 1988; Rosen et al., 1989; Xia & Storm, 1990; Hinz & Kirley, 1988), Lys in three ATP binding domains may not be crucial for interaction with ATP (Table 2), as the case for the transcription termination factor, rho (Dombroski et al., 1988). As discussed in a previous paper (Kimura & Fujisawa, 1991), His553 may not be involved in Mg~+ binding because His553 is not conserved between T3 and T7, although three ATP binding motifs and another Mg2+ binding motif are conserved (Dunn & Studier, 1983; Yamada et al., 1986). As shown in the present paper, gpl9 has multiple ATP and Mgz+ binding motifs in its amino acid sequence, and mutations in these motifs of gpl9 molecules resulted in different defects in DNA packaging process: abortive interactions with ATP, abnormal binding to prohead, loss of the nonspecific and specific endonuclease activities and uncoupling of ATP hydrolysis to DNA translocation. These facts indicate that gpl9 plays crucial roles in DNA translocation and in processing of concatemer DNA. Since the same defects were observed with mutants in different motifs, multiple domains would be implicated in each function. The analyses of the DNA packaging process in the defined systems of T3 and ~b-29 demonstrate that the large subunits, gpl9 ofT3 (Shibata et al., 1987a; Fujisawa et al., 1991) and gpl6 of ~-29 (Guo et al., 1987a), undergo conformation changes in multiple steps of the DNA packaging process through interaction with ATP. In T3, six molecules of gpl9 bind to the connector at the portal vertex to form the functional packaging machinery in the presence of ATP. When gpl9 is bound to proheads, the gpl 9-prohead complex gains the ability to bind to a gpl8-DNA complex in the presence of ATP. ATP binding and hydrolysis in the prohead/gpl9/gpl8/ DNA complex result in DNA translocation into the head. Although speculative models for DNA translocation have been proposed, much attention has not been given to the role of the terminase or packaging enzyme in these models (Earnshaw & Casjens, 1980; Turnquist et al., 1992). In a previous paper (Shibata et al., 1987b), we proposed a ratchet model for DNA translocation in which the gpl9-connector complex located at the portal vertex would translocate DNA into the prohead by a cyclic change in the conformation of gpl9 mediated by ATP. In the model, one molecule of gpl9 is bound to each component of six domains of the connector and one of the gpl9 molecules is bound to the sugar-phosphate backbone of duplex DNA. When ATP is hydrolyzed, gpl9 in the
258
M u t a n t D N A Packaging Protein8 of Phage T 3
compiex contracts, forcing the D N A into the head, and then dissociates from the sBgar-phosphate backbone. A t this m o m e n t , the n e x t g p l 9 binds the neighboring s u g a r - p h o s p h a t e backbone. The same cyclic changes translocate D N A into the head. Ordered conformational changes in proteins t h a t are -driven b y nucleotide t r i p h o s p h a t e hydrolysis provide explanations for A T P - d r i v e n biological machines (Alberts & Miake-Lys, 1992). Rao & Black (1988) proposed a p a c k a s o m e model in which gpl7, the large subt~nit of T4 terminase, constitutes the packaging machinery. According to the model, D N A is translocated into the prohead by a complex structure assembled a t the portal vertex of the prohead utilizing A T P hydrolysis energy. Much remains to be learnt a b o u t the functions of the packaging enzymes or terminases in DNA packaging. We are grateful to Dr Michael Feiss (The University of Iowa) for his invaluable help with the manuscript. This work is supported by a grant-in-aid for scientific research from the Ministry of Education of Japan.
References Alberts, B. & Miake-Lye, R. (1992). Unscrambling the puzzle of biological machines: the importance of the details. Cell, 68, 415-420. Becker, A. & Gold, M. (1988). Prediction of an ATP reactive center in the small subunit, gpNul, of the phage lambda terminase enzyme. J. Mol. Biol. 199, 219-222. Becket, A., Marko, M. & Gold, M. (1977). Early events in the in vitro, packaging of bacteriophage ~ DNA. Virology, 78, 291-305. Berg, J. M. (1986). Potential metal-binding domains in nucleic acid binding proteins. Science, 232, 458-460. Black, L. W. (1989). DNA packaging in dsDNA bacteriophages. Annu. Redv. Microbiol. 43,267-292. Cue, D. & Feiss, M. (1992). Genetic analysis of mutations affecting terminase, the bacteriophage )~ DNA packaging enzyme, that suppress mutations in cos B, the terminase binding site. J. Mol. Biol. 228, 72-87. Davidson, A. R. & Gold, M. (1992). Mutations abolishing the endonuclease activity of bacteriophage 2 terminase lie in two distinct regions of the A gene, one of which may encode a "leucine zipper" DNA-binding domain. Virology, 189, 21-30. Dombroski, A. J., Brennan, C. A., Soear, P. & Platt, T. (1988). Site-directed alterations in the ATP-binding domain of the Rho protein affect its activity as a termination factor. J. Biol. Chem. 263, 18802-18809. Dunn, J. J. & Studier, F. (1983). Complete nucleotide sequence of bacteriophage T7 DNA and the locations of T7 genetic elements. J. Mol. Biol. 166, 477-535. Earnshaw, W. C. & Casjens, S. R. (1980). DNA packaging by the double-stranded DNA bacteriophages. Cell, 21,319-331. Feiss, M., Fogarty, S. & Christiansen, S. (1988). Bacteriophage ). DNA packaging: a mutant terminase that is independent of integration host factor. Mol. Gen. Genet. 212, 142-148. Fraekman, S., Siegele, D. A. & Feiss, M. (1985). The terminase of bacteriophage 2: functional domains for cos B binding and mutlimer formation. J. Mol. Biol. 183, 225-238.
Fry, D. C., Kuby, S. A. & Mildvan, A. S. (1986). ATP-binding site of adenylate kinase; mechanistic implications of its homology with ras-encoded p21, FI-ATPase, and other nucleotide-binding protein. Proc. Nat. Acad. Sci., U.S.A. 83,907-991. Fujisawa, H. & Yamagishi, M. (1981). Studies on factors involved in in vitro packaging of phage T3 DNA. Progr. Clin. Biol. Res. 64, 239-252. Fujisawa, H., Miyazaki, J. & Minagawa. T. (1978). In vitro packaging of phage T3 DNA. Virology, 87, 394-400. Fujisawa, H., Kimura, M. & Hashimoto, C. (1990). In vitro cleavage of the concatemer joint of bacteriophage T3 DNA. Virology, 174, 26-34. Fujisawa, H., Shibata, H. & Kato, H. (1991). Analysis of interactions among factors involved in the bacteriophage T3 DNA packaging reaction in a defined in vitro system. Virology, 185, 788-794. Guo, P., Petersen, C. & Anderson, D. (1987a). Initiation events in in vitro packaging of bacteriophage ~b29 DNA-gp3. J. Mol. Biol. 197, 219-228. Guo, P., Peterson, C. & Anderson, D. L. (1987b). Prohead and DNA-gp3-dependent ATPase activity of the DNA packaging protein gpl6 of bacteriophage ~b-29. J. Mol. Biol. 197, 229-236. Hamada, K., Fujisawa, H. & Minagawa. T. (1986a). Overproduction and purification of the products of bacteriophage T3 genes 18 and 19. two genes involved in DNA packaging. Virology, 151, 110-118. Hamada, K., Fujisawa, H. & Minagawa, T. (1986b). A defined in vitro system for packaging of bacteriophage T3 DNA. Virology, 151, 119-123. Hamada, K., Fujisawa, H. & Minagawa, T. (1987). Characterization of ATPase activity of a defined in vitro system for packaging of bacteriophage T3 DNA. Virology, 159, 244-249. Hashimoto, C. & Fujisawa, H. (1988). Packaging and transduction of non-T3 DNA by bacteriophage T3. Virology, 166, 432-439. Higgins, R. R., Lucko, H. J. & Becker, A. (1988). Mechanism of cos cleavage by bacteriophage A terminase: multiple roles of ATP. Cell, 54, 765-775. Hinz, H. R. & Kirley, T. L. (1990). Lysin 480 is an essential residues in the putative ATP site of lamb kidney (Na, K)-ATPase. J. Biol. Chem. 265, 10260-10265. Kimura, M. & Fujisawa, M. (1991). Dissection of functional domains of the packaging protein of bacteriophage T3 by site-directed mutagenesis. Virology, 180, 709-715. Kunkel, T. A., Roberts, J. D. & Zakour, R. A. (1987). Rapid and efficient site-specific mutagenesis without phenotypic selection. In Methods in Enzymology (Wu, R. & Grossman, L. eds), vol. 154, pp. 367-382. Academic Press, San Diego. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-685. Maruyama, K. & MacLennan, D. H. (1988). Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca 2+ transport activity of Ca2+-ATPase expressed in COS-cells. Proc. Nat. Acad. Sci., U.S.A. 85, 3314-3318. Morita, M., Tasaka, M. & Fujisawa, H. (1993). DNA packaging ATPase of bacteriophage T3. Virology 193,748-752. Muriaido, H. & Becker, A. (1987). Head morphogenesis of complex double-stranded DNA bacteriophages. Microbiol. Rev. 42, 529-576.
Mutant DNA Packaging Proteins of Phage T3 Nakasu, S., Fujisawa, H. & Minagawa, T. (1983). Role of gene 8 product in morphogenesis of bacteriophage T3. Virology, 127, 124-133. Parris, W., Davidson, A., Keeler, C. L. Jr. & Gold, M. (1988). The Nul subunit of bacteriophage ). terminase. J. Biol. Chem. 263, 8413-8419. Rao, V. B. & Black, L. W. (1988). Cloning, overexpression and purification of the terminase proteins gpl6 and gpl7 of bacteriophage T4. Construction of a defined in vitro DNA packaging system using purified terminase proteins. J. Mol. Biol. 200, 475-488. Rozen, F., Pelletier, J., Trachsel, H. & Sonenberg, N. (1989). A lysin substitution in the ATP-binding site of eukaryotic initiation factor 4A abrogates nucleotide-binding activity. Mol. Cell. Biol. 9, 4061-4063. Shibata, H., Fujisawa, H. & Minagawa, T. (1987a). Early events in DNA packaging in defined in vitro system of bacteriophage T3. Virology, 159, 250-258. Shibata, H., Fujisawa, H. & Minagawa, T. (1987b). Characterization of the bacteriophage T3 DNA pack-
259
aging reaction in vitro in a defined system. J. Mol. Biol. 196, 845-851. Turnquist, S., Simon, M., Egelman, E. & Anderson, D. (1992). Supercoiled DNA wraps around the bacteriophage ~b29 head-tail connector. Proc. Nat. Acad. Sci., U.S.A. 89, 10479--10483. Xia, Z. & Strom, D. R. (1990). A-type ATP binding consensus sequences are critical for the catalytic activity of the caimodulin-sensitive adenylyl cyclase from Bacillus anthracis. J. Biol. Chem. 265, 6517-6520. Yamada, M., Fujisawa, H. & Minagawa, T. {1986). Isolation and characterization of bacteriophage T3fr7 hybrids and their use in studies on molecular basis of DNA packaging specificity. Virology, 144, 502-515. Yanisch-Perron, C., Vieria, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene, 33, 103-119.
Edited by N. Sternberg (Received 19 A p r i l 1993; accepted 24 August 1993)
Analysis of Functional Domains of the Packaging Proteins of Bacteriophage T3 by Site-directed Mutagenesis Miyo Morita, Masao Tasaka and Hisao Fujisawat Department of Botany, Faculty of Science Kyoto University, Kyoto 606, Japan
Intracellular phage T3 DNA is synthesized as a concatemer in which unit-length molecules are jointed together in head-to-tail fashion through terminally redundant sequences. The concatemeric DNA is processed and packaged into the prohead with the aid of non-capsid proteins, gpl8 and gpl9. We have developed a defined system, composed of purified gpl8, gpl9 and proheads, and a crude system, composed of lysates of T3 infected cells, for in vitro packaging of T3 DNA. The defined system displays an ATPase activity which is composed of DNA packaging-dependent and -independent ATPases (pac- and nonpac-ATPases, respectively). In the crude system, DNA is packaged by a way of concatemer as an intermediate, gpl9 has ATP binding activity and three ATP binding and two Mg2+ binding consensus motifs in its amino acid sequence. We have expanded the previous studies on the roles of these domains in the DNA packaging reaction by more extensive analysis by sitedirected mutagenesis, gpl9 mutants, including the previously isolated four mutants, were divided into four groups according to the DNA packaging ativity in the defined and crude systems: group 1 mutants were defective in both systems (gpl9-G61D, which is a gpl9 mutant with Gly to Asp at amino acid 61 and so on, and gp19-H344D); the group 2 mutant had decreased activity in both systems (gp19-G429R); group 3 mutants were active in the defined system but defective in the crude system (gp19-G63D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-G424E); group4 mutants had almost the same activity as gpl9-wt (gp19-K64T, gp19-K370I, gp19-G429L, gp19-K430T and gp19-H553L). Group 1 mutants had an altered conformation, resulting in defective" interaction with ATP and in abortive binding to the prohead, and lost specifically the pac-ATPase activity. The group 2 mutant had an increased pac-ATPase activity in spite of the decreased DNA packaging activity, indicating that this mutant is inefficient in coupling of ATP hydrolysis to DNA translocation. The inability of the group 3 mutants except gp19-H347R to package DNA in the crude system would be due to a defect in processing of concatemer DNA. gp19-H347R would be a mutant defective in the initiation event(s) of DNA packaging.
Keywords: T3 phage; DNA packaging protein gpl9; site-directed mutagenesis; functional domains
1. I n t r o d u c t i o n
jointed together in a head-to-tail fashion through the terminally redundant sequences. During packaging of DNA, mature monomers are cut from the concatemer. DNA packaging is thought to proceed by a common mechanism for most dsDNA phages (Murialdo & Becker, 1978; Earnshaw & Casjens, 1980). Formation of precursors for DNA translocation, prohead/packaging enzyme/DNA complexes, depends upon either ATP as an allosteric effector (T3; Shibata el al., 1987a) or ATP hydrolysis (~t; Becket et al., 1977; 429; Guo et al., 1987a). DNA translocation into the prohead is driven by ATP hydrolysis (Black, 1989). Packaging
During head assembly of most double-stranded (ds:~) DNA bacteriophages, DNA is packaged into the cavity of a preformed protein shell, called the prohead, with the aid of non-capsid protein, called a terminase or packaging enzyme. DNA is synthesized as concatemers, in which unit-length molecules are t Author to whom all correspondence and reprint requests should be addressed. :~ Abbreviations used: ds, double-stranded; gp, gene product; wt, wild-type; peq, phage equivalents. 0022-2836/94/010248-12 $08.00/0
248 © 1994 AcademicPress Limited
Mutant D N A Packaging Proteins of Phage T3 enzymes (terminases) and in vitro packaging systems display a DNA dependent ATPase activity. These results indicate that ATP plays multiple roles in the DNA packaging reaction. In general, the packaging enzyme is commonly composed of two subunits; the small subunit has DNA binding activity and the large subunit has prohead binding activity. It is thought that the large subunit carries the crucial functions, whereas the small subunit is stimulative for packaging of mature DNA (Black, 1989). In general, the large subunits have the ATP binding and potential Mg2+ binding motifs (Guo et al., 1987b). In the case of 2, the small subunit also has ATP binding motifs (Becket & Gold, 1988) and displays a DNA independent ATPase (Parris et al., 1988). To elucidate the DNA packaging mechanisms at the molecular level, we have constructed a defined and a crude in vitro system for packaging phage T3 DNA. The crude system contains extracts of T3-infected cells and DNA is packaged by way of concatemer as an intermediate (Fujisawa et al., 1978). The defined system is composed of mature T3 DNA, purified proheads and two packaging proreins, gpl8 and gpl9, corresponding to the small and large subunits of the packaging enzyme, respectively (Hamada et al., 1986b). Our previous results indicate the sequential events in DNA packaging: the packaging proteins gpl8 and gpl9 bind DNA and proheads, respectively (Shibata et al., 1987a). These complexes associate to form a direct precursor complex (50 S complex) for DNA translocation into the head. The formation of the gpl9-prohead complex and the 50S complex requires ATP as an allosteric effector. The DNA translocation into the head is driven by hydrolysis of ATP. The defined in vitro DNA packaging system displays an ATPase which is composed of DNA packaging:dependent and -independent ATPases (pac- and nonpac-ATPases, respectively: Morita et al., 1993).
gpl9 is only an ATP-binding protein in the defined packaging system (Hamada et al., 1987) and has putative ATP and Mg2+-binding consensus sequences (Fig. 1 and Table 1; see Guo et al., 1987b). Therefore, it is expected that gpl9 is involved in ATP interaction in formation of the gpl9-prohead and the 50 S complexes, and in ATP hydrolysis in DNA translocation. Previously, we have undertaken a molecular genetic approach to dissect functional domains of gpl9 by site-directed mutagenesis into these ATP and metal binding motifs, and mutant proteins were partially characterized. Two important mutants were isolated: mutant gpl9 with a Gly to Asp mutation at amino acid 61 (gpl9-G61D) was defective in DNA packaging, probably due to an altered interaction with ATP; and gp19-G424E, with a mutation in another putative ATP binding domain, was active in DNA packaging but defective in cutting of the concatemer (Kimura & Fujisawa, 1991). These results show that the ability to inactive function(s) of gpl9 by single amino acid substitution is useful in studies to understand the mechanisms of the DNA packaging in which gpl9 plays a central role. In this paper, we have expanded the previous studies on gpl9 by more extensive analysis by sitedirected mutagenesis to define the role(s) of large subunit gpl9 in the packaging machinery. The results suggest that the ATP binding domain-I (Gly61) and Mg2+ binding domain-I (His344) are involved in the gpl9-ATP interaction, and that the ATP-binding domain-III (Gly429) is important in coupling of ATP hydrolysis to DNA translocation gpl9 is responsibile for processing of concatemers in concert with multiple ATP-binding domains. 2. M a t e r i a l s a n d M e t h o d s
(a) Bacteria, phages and plasmids Escherichia coli CJ236 (dut 1 ung 1 thi 1 ,'el AI; pCJl05 (Cmr)) was used for preparation of M13 viral DNA
Table 1 Comparison of A T P binding sequences of the T3 D N A packaging protein gp19 and other proteins
Proteins
Residues
T3 gpl9
52-72 360-372 484-505 154-174 293-314 164-184 60-80
2gpA T4 gpl7 DnaA RecA ATPase = of E. coil Myosin of rabbit
164-184
173-192
T3 gpl9 Bovin PKA RSV p60"re
418-435 44-62 269-287
EGF receptor FSV ==='rp~
690-707 923-941
249
Amino acid sequences KKF-ILQAFRGIGKSFITCAFVV ILVIDPSGRGKDE RVI-PIKGASVYGKPVASMPRKR _RMTVCNL_S-RQLGKTT-VVAIFL HFY-DIWTAAVEGKSGFEPYTAI Y_NPLFLYGGTGLGKTT-FAMNLV _R-IVEIYG PGSSGKTT-LTLQVI _R--ELIIGDRGTGKTALAIDAII _S--ILITGESGAGKTV-NTKKVI VFESNFGDGMFGKVFSPVL ERIKTLGTGSFGRVMLVKH RLEAKLG_G_Q_GCFGEVWMGTW KI-KVLGSGAFGTVYKGLW LLGERIGRGNFGEVFSGRL
Residues showing substantial homology between gpl9 and other ATP binding proteins are underlined.
M u t a n t D N A Packaging Proteins of Phage T 3
250 0
containing uracil (Kunkel et al., 1987). E. coli JMl03 was used as a host (Ung+~ for most of the p~asmid constructs derived from MI3 m p l 9 (Yanish-Perron et al., 1985). A host for expression vector pNT45, which uses ). PL promoter for expression of cloned genes, is described in a previous paper (Hamada et al., 1986a). Amber mutants of genes 3 (endonuclease, 3-), 5 (DNA polymerase, 5-) and 19 (a large subunit of packaging proteins, 19-) of T3 phage were from our laboratory stocks, p K H 2 is a pNT45 derivative carrying a 2"32 kb fragment containing gene 19, which is an expression vector for gene 19 (Hamada et al.~ 1986a). pUC18 E1-TR is pUCl8 carrying an insert of about 500 bp t h a t includes the terminally redundant sequence and its flanking sequences from T3 eoneatemeric DNA (TR sequence), plus 3-1 kb including gene 19 (El sequence: Hashimoto & Fujisawa, 1988). Plasmid was iinearized by PstI digestion. (b) Buffers Buffer PM is l0 mM potassium phosphate (pH 7-4) with 7 mM 2-mercaptoethanol; buffer PMG is a buffer PM with 5 % (v/v) glycerol. Complete pac buffer is 5 % (w/v) polyethylene glycol (M, 6000), 100/~M ATP, 20 mM Tris" HCI (pH 7"4), 50 mM NaCl, 1 mM spermidine, 5 mM MgC! 2, 7 mM 2-mercaptoethanol. Prohead buffer is described in a previous paper (Nakasu et al., 1983).
an Applied Biosystems 3804B-02 DNA synthesizer. Each oligonucleotide carried l or 2 mismatched nucleotides that created both an amino acid substitution and a new restriction site at the target region {Fig. l(B)). The termini of oligonuc[eotides were phosphorylated by T4 polynucleotide kinase. M13 m p l 9 viral DNA that carried gene 19 was propagated in E. coil CJ236. To this viral DNA, containing uracil, oligonucleotides were hybridized. and extended with the use of T4 DNA polymerase. T4 DNA ligase was used to close the stands, and the products were introduced into E. coli JMl03 cells. The resulting plaques were isolated and mutants were screened by analysis of newly generated restriction sites. The mutation sites were confirmed by DNA sequencing. (f) In vitro D N A packaging reaction in the defined system A standard reaction mixture (20pl) contained l0 I° phage equivalents (peq) of mature T3 DNA or 0"5 pg DNA, 4 x l0 l° peq of proheads, 20pmol of g p l 8 and 3 pmol of g p l 9 in complete pac buffer (Shibata et al., 1987). After the reaction, the mixture was treated with DNase I (0-2 mg/ml) for 20 min on ice to digest unpackaged DNA, if necessary, and mature heads were converted to infectious particles by incubation with a head acceptor extract containing tail and tail fiber proteins, and phages were assayed by titration on E. coli BB as described (Shibata et al., 1987). Alternatively, DNA in the head was subjected to agarose gel electrophoresis as described in a previous paper (Shibata et al., 1987).
(c) Purification of phage proteins involved in
D N A packaging Proheads and g p l 8 were purified as described in a previous paper (Hamada et al., 1980a). (d) Purification of 9p19 g p l 9 s were purified according to Kimura & Fujisawa (1991). However, some m u t a n t gpl9s (gp19-K64T and gplg-H344D), which were incorporated into inclusion bodies, were purified as following. Cells from 2 1 of overexpressed culture were suspended in 20 m] of PM buffer containing 2 mM EDTA and frozen at - 8 0 ° C . Frozen cells were thawed at 30°C, mixed with 2 ml of lysozyme (10 mg/ml) and kept on ice for 1 h. After addition of 2-5 ml of 5 M NaCI and 0-005 ml of l M MgCI 2, the lysate was sonieated and centrifuged at 8000revs/min in a sorval SS-34 rotor for 15 min. The pellet was suspended in 15 ml" buffer PMG, sonicated and centrifuged as above. The pellet was suspended in 2 ml buffer PMG and combined with 18 ml of 7 M guanidine" hydrochloride in 20 mM phosphate buffer (pH 7"4), l mM DTT. After 30 min on ice, the mixture was dialyzed against I I of 0-2 M NaC1, l mM MgCI 2 in buffer PMG for 6 h and then against I I of 0"1 M NaCl, 1 mM MgC12 in buffer PMG overnight. The lysate was centrifuged to remove aggregates. The supernatant was loaded onto a DEAE-eellulose column (DE52, 5 ml), equilibrated with 0"l M NaCI in buffer PMG, and eluted by a gradient of 0"l M to 0"3 M NaCI in buffer PMG. The procedure hereafter was the same as t h a t for purification of wild-type g p l 9 (Kimura & Fujisawa, 1991).
(e) Oligonucleotide-directed mutagenesis Site-directed mutagenesis was carried out according to the protocol of Kunkel et al. (1987) (Kimura & Fujisawa, 1991}. Primers (33 to 36 bases long) were synthesized by
(g) In vitro D N A packaging in the crude system The reaction mixture contained g p l 9 and 19- extract, a mixture of 3 - / 1 9 - and 5 - / 1 9 - extracts prepared as described elsewhere (Fujisawa & Yamagishi. 1981). supplemented with 2 mM ATP. After the addition of T3 wild-type DNA, the mixture was incubated for 60 min at 30°C. The reaction was stopped by adding chloroform, and after appropriate dilution, phages were titrated on E. coli ER22 (Fujisawa et al., 1978). (h) Isolation of gp19-prohead complexes g p l 9 and proheads were incubated in complete pac buffer with or without ATP at 30°C for 30 min at a molar ratio of g p l 9 to a prohead of 40. After incubation, the reaction mixture was sedimented through 2 ml of 10% (w/v) sucrose in pac buffer with or without ATP in an Hitachi RPS50 at 26,000 revs/min for 90 rain. Pellets were dissolved in sample buffer and subjected to SDS/polyacrylamide gel electrophoresis (Laemmli, 1970). (i) Assay of ATPa~es For ATPase assay, the same standard reaction mixture for in vitro DNA packaging in the defined system as described above was used. ATPase assays were performed as described in a previous paper (Morita et al., 1993). Briefly, the reaction mixture containing [y-32P]ATP at 1-5MBq/ml (100/~M) was incubated with or without actinomycin D (l juM) for 30min at 30°C. Two pl of the reaction mixture was spotted onto PEI-cellulose (Maehery-Nagel. Co.) and dried. The chromatogram was soaked in methanol for 5 min, redried and run in I M formic acid, 0"5 M LiCI. The distribution of radioactivity on ATP and Pi was determined by an image analyzer (Ambis Image Analyzer). The pac- and nonpac-ATPase
Mutant DNA Packaging Proteins of Phage T3
A)
251
100
20O
3O0
400
500
I
I
I
I
I
ml i
I
t
I ATP binding I HLQAFR~IQ._KSF G61D G63D K64T
Mg -binding I _HSYHSC H344D H347R
T
c
586
m I
ATP binding HI ATP binding II ILVIDPSGR.Q_KDE NF~MFfi.K~ G424E G367D G429R G369D G429L K370I K430T
TI Mg -binding II
HM~tP[H I-I553L
B) Restriction enzyme 369 370 367 wild type 5'- CCT AGT GGT CGA GGT AAG GAT -3' P S G R G K D G367D
~ ~ 3 A S D R
G369D G
R
D
Nru l
Figure 1. Site-directed mutagenesis of gpl9. (A) The relative positions of ATP binding and Mg2+ binding motifs and mutated amino acids, which are underlined. Amino acid residues were numbered from the intiation Met (1) predicted from DNA sequences (Yamada et al., 1986). (B) Examples of amino acid substitution and new restriction enzyme sites (boxed) created by nucleotide substitutions (arrow heads).
activities were a difference between total and actinomycin D-resistant ATPases and the actinomycin D-resistant ATPase. respectively. 3. Results
(a) Construction of amino acid substitution mutants Starting from pKH2, the 2-32 kb region, carrying the gene 19, was inserted into the BamHI-EcoRI region in the multicioning site of M13 mpl9. To the resulting construct, the synthetic oligonucleotides were hybridized, and olgonucleotide-directed routsgenesis was carried out by the method of Kunkel et al. (1987). The sequences of the oligonucleotides were designed not only to replace a single amino acid but also to create a new restriction site in the ATP binding and Mg2+ binding domains (Fig. I(B)), as described in a previous paper (Kimura & Fujisawa, 1991). This made it easy to screen for the amino acid substitution mutants, and nine mutants were newly isolated in the present paper. Hereinafter, gpl9 of the wild-type and its derivatives carrying mutations described above are abbreviated as gpl9-wt and gp19-G63D, etc.
after induction by temperature shift-up, proteins expressed from the cloned genes were identified through SDS/polyacrylamide gel electrophoresis. In addition to the previous four mutants (gpl9-G61D, gp19-G424E, gp19-K430T and gp19-H553L: Kimura & Fujisawa, 1991), nine mutants (gplgG63D, gp19-K64T, gp19-H344D, gp19-H347R, gp19-G367D, gp19-G369D, gp19-K370I, gp19G429L and gp19-G429R) were purified to near homogeneity by the standard method (Kimura & Fujisawa, 1991). However, two mutant proteins (gp19-K64T and gp19-H344D) were incorporated into inclusion bodies when overexpressed. These mutant proteins were solubilized by guanidinehydrochloride from inclusion bodies and purified by the standard procedure as described in Materials and Methods. gpl9-wt was also incorporated into including bodies by a longer incubation at 42°C, gpl9-wt purified from inclusion bodies had the same specific activities of DNA packaging in the defined and crude systems as gpl9-wt purified by the standard method (data not shown).
(c) D N A packaging activity (b) Overexpression and purification of 9ene product First, the 2"32 kb fragment was generated from the M13 clones by Sau3A-EcoRI digestion and inserted into the BamHI and EcoRI sites of the expression vector pNT45 in the proper orientation (Hamada et al., 1986a). The resulting constructs were introduced into ER22 (pNT203) cells, and
To assay DNA packaging activity, gpl9-wt or mutant was added to a crude or a defined in vitro system for DNA packaging and assayed by measuring plaque-forming units as described in Materials and Methods. As shown in Table 2, gpl9-G61D, gp19-G63D, gp19-H344D, gp19-H347R, gp19-G367D, gpl9-
Mutant DNA Packaging Proteins of Phage T3
252 Table 2
D N A packaging in the defined ,and crude in vitro systems Packaging activity (% of WT) gpl9
Crude system
Defined system
0' 16 100 0"054 0"47 96"7 3'9 l'21 0"47 1"72 65"8 2"6 35"5 27"3 69'6 126
0"00 100 0"4 25"l 104 l "7 55"2 23"2 77'0 96"1 121"6 98"3 16'3 65"9 110-5
-WT G61D G63D K64T H344D H347R G367D G369D K370I G424E G429L G429R K430T H553L
(e) Specific cleavage activity
DNA packaging reaction was performed by using the defined and crude in ~itrosystems as described in Materials and Metbods. G369D and gp19-G424E were defective in DNA packaging in the crude in vitro system. In the defined system, gpl9-G61D and gp19-H344D were defective, gp19-G63D, gp19G367D and gp19-G429R had decreased activity, and the other m u t a n t s had almost the same activity as gpl9-wt. Accordingly, gp19-G63D, gp19-H347R, gp19-G367D and gp19-G369D were defective in DNA packaging in the crude system but active in the defined system as previously observed with gp19-G424E ( K i m u r a & Fujisawa, 1991). (d) Non-specific endonuclease activity g p l 9 - w t has a non-specific endonuclease activity, suppressible by either A T P or gpi 8 (Fujisawa et al., 1990). To examine the endonuclease activity of m u t a n t gpl9s, circular pUCI8 E I - T R was incu-
~
ATP
+ ' -
+
-
+
bated with or without ATP. Mutants defective in the crude system but. active in the defined system (gp19-G63D, gp19-367D, gpi9-G369D), except gp19-H347R, lacked the endonuclease activity (Fig. 2). Other m u t a n t s showed almost the same activity as gpl9-wt, except t h a t the endonuclease activity of gpl 9-H344D was not suppressed by A T P (data not shown) as is t h a t of gplg-G61D ( K i m u r a & Fujisawa, 1991).
In the defined in vitro I)NA packaging system. linearized plasmid DNAs carrying the concatemer junction are specifically cleaved at the left end of the terminally r e d u n d a n t sequence when packaged leftward, indicating t h a t the cleavage reaction corresponds to the termination cleavage of concatemer processing (Fujisawa et al., 1990). As shown in Figure 3, the products in the cleavage reaction with g p l 9 - w t were a fragment of 3"0 kb, containing the terminally r e d u n d a n t sequence and the remainder of the plasmid, a 2"5 kb fragment, and both fragments were protected from DNase I digestion as shown in a previous paper (Kimura & Fujisawa, 1991). The same results were obtained with gp19-K3701, gp19-G429R, gp19-G429L and gp19-K430T. When the reaction was carried out with m u t a n t s defective in the nonspecific endonuclease activity (gp19-G367 D, gp19-G369D and gp19-G424E), the cleavage was not detected, but when the reaction mixtures were treated with DNase I to digest unpackaged I)NA the 3-0kb fi'agment containing the terminally r e d u n d a n t sequence appeared, d e m o n s t r a t i n g that DNA was packaged leftward and stopped at the left end of the terminally r e d u n d a n t sequence without being cleaved. With gp19-H344D, the cleavage was not. detected and all DNA was digested by DNase I (data not shown), as observed with gpl9-G611) (Kimura & Fujisawa, 1991), d e m o n s t r a t i n g t h a t DNA was not packaged.
r ~.
-
+
-
~
ATP
+
~
-
+
~
-
+
-
+
i
linear
--
lmem"
Figure 2. Endonuelease activity ofgpl9. Closed circular pUCI8 EI-TR DNA w a s incubated with gpl9 in the presence (+) or absence ( - ) of ATP in pac buffer at 30°C for 60 min and subjected to agarose gel electrophoresis. The lmsition of closed circular tee), nicked circular (nc) and linear forms of pUC EI-TR are indicated.
Mutant DNA Packaging Proteins of Phage T3 A
-DNaseI
+DNasel
-DNaseI
253 +DNaseI
N
,
~
~
4730bp! 2920bp 2580bp 2080bp
Cleavage
B
Psi11
W 2.5kb
~l~ I~
3.0kb
Pstl I
~[
direction of packaging Figure 3. Cleavage of the coneatemer joint in the defined in vitro DNA packaging system. (A) Linearized pUCI8 EI-TR was ineubated in the standard reaction mixture containing 250/~M ATP at 30°C for 60 min and sample buffer or 1)Nase I was added. Samples with DNase I were incubated on ice for 20 rain before addition of sample buffer. DNAs were then subjected to agarose gel eleetrophoresis as described in Materials and Methods. Sizes of the molecular weight standards are indicated. (B) A diagram of the packaging and cleavage reaction. The direction of DNA packaging and a site of I)NA cleavage are indicated by arrows.
(f) ('haracterization of gp19 mutants in the D N A packaging reaction in the defined in vitro system |)uring DNA packaging in the defined system, gpl9 and gpl8 form separate complexes with proheads and DNA, respectively. These two complexes form a 50 S complex and then DNA translocation is driven by hydrolysis of ATP. The formation of the g p l 9 - p r o h e a d and the 50S complex depends upon ATP as an allosteric effector (Shibata et al., 1987a). To characterize the defect in DNA packaging of the group 1 mutants, the g p l 9 - p r o h e a d interaction was analyzed by determining copy numbers of gpl9 in g p l 9 - p r o h e a d complexes, g p l 9 - p r o h e a d complexes were isolated and subjected to SDS/ polyacrylamide gel electrophoresis as described in Materials and Methods. Relative band densities of gpl9 to gpl8 of Coomassie brilliant blue-stained gel were calibrated by a densitometer to estimate copy numbers in the complexes as described in a previous paper (Fujisawa et al., 1991). As shown in Figure 4, we confirmed the previous results that gpl9-wt bound to a prohead at molar ratios of 6 or 20 in the presence or absence of ATP, respectively (Fujisawa et al., 1991). However, gpl9-G61D bound to a prohead at a molar ratio of 20, independent of the presence or absence of ATP. gpl 9-H344D bound to a prohead at ratios of more than 6 to 20, indepen-
dent of the presence or absence of ATP (data not shown). These results indicate that group 1 mutants are abnormal in prohead binding activity. To characterize gpl9 mutants, the effect of gpl9 mutation on the rate of DNA translocation was examined. The defined standard reactions were incubated in the presence of ATP-y-S at 30°C for five minutes to allow formation of the precursor 50 S complex; DNA packaging was initiated at 20°C by diluting the reaction mixture 100 times into complete pac buffer containing ATP. The reaction was terminated by the addition of DNase I and mature heads were assayed as described in Materials and Methods. With all mutants, the kinetics of the appearance of mature heads was not different from that of gpl9-wt, indicating that the DNA translocation rate was not affected by gpl9 mutants (data not shown). However, with gp19-G429R and gpl9-G429L (Fig. 5), the conversion of most 50 S complexes into mature heads took a much longer time, and with gp19-G429R mature head production decreased two orders of maginitude when started from the 50 S complex.
(g) Tryptic digestion of mutant gp19 The cleavage patterns of gpl9-wt by several proteases were altered by the addition of ATP,
254
Mutant DN.4 Packaging Proteins of Phage T3
f~
!
WT II +
+ATP
G61D
I -
I
+
•
WT
ATP p
', K64T
' G61D ' G63D
I 2 3 4 511 2 3 4 5 1 1 2 3 4 511 2 3 4 5
gpl6 gpl5 - - gpl9
67kl)
' - x ~, 8
45kD 39kD
gp9, gpl0
'i
gpl0
E Trypsin
(a)
-- ATP u
WT
~, G61D
i 2 3 4 5',1 2 3
~:'~::-~-~-~ ~ : ~=*~*"< ., "
'~ G63D ~ K64T
4 5',12 3 4
s',~ 2 3
u
4 5 r-
' *~i:'~:. *;~~~'~ --67kD
Figure 4. SDS/polyaerylamide gel electrophoresis of gpl9-prohead complexes. Mixtures containing proheads and 40 molecules per prohead of gpl9-wt or gpl9-G61D in pac buffer were incubated in the presence ( + ) or absence ( - ) of ATP at 30°C for 30 min. gplg-prohead complexes were prepared as described in Materials and Methods and subjected to SDS/polyacrylamide gel electrophoresis.
--45kD --39kD
i (b)
0 8
H344D
WT I
I
I I -ATP
+ATP
-ATP
M I 2 3 4 5 2 3 4 5 I 23
÷ATP
4 5 2 345
M 97.4kl)
~"
6-
66.2k1)
45kl)
5"
31kD
0
10
20
Time (rain) Figure 5. Time-course of formation of mature heads from the 50 S complex. A standard reaction containing ATP-7-S in place of ATP was ineubat~d at 30°C for 5 min to allow formation of the 50 S precursor complex and then diluted I00 times into the complete pac buffer containing ATP at 20°C to initiate DNA transloeation into the head. The reaction was stopped by the addition of DNase I at the indicated times (min) and mature beads were assayed as described in Materials and Methods. Wild-type (O); G429L (0); G429R ( , ) . P.F.U., plaque-forming units.
21.5kl)
(c)
Figure 6. Effect of ATP on the cleavage of gpl9 by trypsin, gpl9 (2-5 g/20/~l) was exposed in the presence or absence of ATP (50 FM) to 2/~g/20 Fl or 0"l Fg/20 Fl of trypsin, respectively, for various times at 20°C (lanes l, 0 rain; 2, 5 min; 3, l0 rain; 4, 30 rain; 5, 90 rain). Portions were subjected SDS/polyacrylamide gel electrophoresis.
Mutant DNA Packaging Proteins of Phage T3 indicating that ATP induces a conformational change in gpl9 (Fujisawa et al., 1991). The functional change(s) induced by a mutation might be related to a change(s) in the conformation of gpl9, reflecting an altered sensitivity to proteases. To examine the protease sensitivity of gpl9 mutants, gpl9s were incubated in the presence or absence of ATP at 20°C and subjected to limited digestion with trypsin at 20°C. As shown in Figure6(A) and (B), the digestion pattern of gpl9-wt by trypsin was affected by the addition of ATP as described in a previous paper (Fujisawa et al., 1991). gpl9-G61D and gp19-H344D (Fig. 6(A), (B). and (C)) were more sensitive to trypsin than gpl9-wt. Their digestion patterns were the same as each other and different from that of gpl9-wt but not significantly affected by the addition of ATP. gp19-G63D, gp19-K64T (Fig. 6(A) and (B)) and other gpl9 mutants (data not shown) were indistinguishable from gpl9-wt in their sensitivity to trypsin, the digestion patterns and the effect of ATP on the digestion patterns. (h) pac-ATPase activity As shown in a previous paper (Hamada et al., 1987), the defined in vitro DNA packaging system displays a DNA-dependent ATPase activity. But the ATPase activity is stimulated by non-packageable DNA such as single-stranded or circular DNA or even RNA (nonpac-ATPase). Among inhibitors of DNA packaging, actinomycinD specifically inhibited the ATPase, strictly coupled to DNA translocation into the prohead (pac-ATPase). The
Table 3 pac- and nonpac-ATPase activities of the defined in vitro system 32p-ATP hydrolyzed(nmol/ml)
gp|9
~n-p~-ATPase
pac-ATPase
WT ATP-binding I G61D G63D K64T Mg"+-binding I H344D H347R ATP-binding II G367D G369D K370I ATP-binding III G424E
27"8+4"2
17'6+ 1-6
19.0+3.1 N.D. N.D.
1'6+0-6 N.D. N.D.
24"0+ 3"8 N.D.
2-04-0"5 N.D.
24-2+4"6 22"0+3"5 68"0+10-1
13"0+2"1 13"2-t-1"1 17"0+1"9
25"6+ 5'2
G429L G429R K430T Mg2 +-binding
24"2 4- 4"2 27"4 4- 5"0 42"2 4- 6"2
16"64-1"8 25"4 4- 2"3 24-2 + 3"3 17"6_+_1"8
N.D.
N.D.
H533L
II
The pac- and nonpac-ATPase activities of the defined in vitro system were measuredas described in Materialsand Methods.
255
pac-ATPase activity is, therefore, defined as the difference between total and actinomycin D-resistant, nonpac-ATPases (Morita et al., 1993). To examine the effect of gpl9 mutations on pacand nonpac-ATPase, the ATPase activities were determined in the presence or absence of actinomycin D. As shown in Table 3, gplg-G61D and gp19-H344D, defective in DNA packaging in the defined system, lacked pac-ATPase activity, although the nonpac-ATPase activity was not affected, gp19-G429R had an increased pac-ATPase activity, in spite of the decrease in the DNA packaging activity. There was no mutant defective in the nonpac-ATPase, although gp19-K370I and gp19430T had an increased activity of the nonpacATPase.
4. D i s c u s s i o n
As observed among several dsDNA phages, the packaging enzyme is commonly composed of two subunits. The small is implicated in DNA binding and stimulates DNA packaging. The large subunit binds to the prohead and appears to be crucial for DNA packaging (Black, 1989). In general, the large subunit has consensus sequences for ATP binding and potential Mg 2+ binding regions (Guo et al., 1987b). Analysis of DNA packaging in the defined system of T3 phage indicates that ATP has multiple functions in DNA packaging as an allosteric effector in formation of two precursor complexes, the gpl9-prohead complex and the 50 S (prohead/packaging enzyme/DNA) complex, and as an energy source in DNA translocation into the head (Shibata et al., 1987a,b). In ). (Becker et al., 1977) and ~b-29 (Guo et al., 1987a), the intermediates in the DNA packaging process are unstable in the presence of non-hydrolyzable ATP analogs and are stabilized by ATP hydrolysis. A necessary approach to understanding the molecular mechanism of DNA translocation driven by ATP hydrolysis is the characterization of interaction between ATP and the packaging machinery, gpl9, the large subunit of the T3 packaging enzyme, is only an ATP binding protein in the packaging system (Hamada et al., 1987) and ATP induces a conformational change in gpl9 (Fujisawa et al., 1991). gpl9 carries ATP and Mg2+ binding motifs. A series of mutations were introduced into both ATP and Mg2+ binding motifs of gpl9 and analyzed. Table 4 summarizes properties of gpl9 mutants, including the previously isolated four mutants (Kimura & Fujisawa, 1991). gpl9 mutants were divided into four groups according to the DNA packaging activity in the defined and crude systems (Tables 2 and 4): group 1 mutants were defective in both systems (gplg-G61D, gplg-H344D); a group 2 mutant had decreased activity in both systems (gplg-G429R); group 3 mutants were defective only in the crude system (gp19-G63D, gplg-H347R, gpl9-G3fi7D, gp19-G369D, gp19-G424E); group4 mutants had almost the same activity as gpl9-wt
Mutant DNA Packaging Proteins of Phage T3
256
Table
4
Summary of the properties of mutant gp19s Packaging activity
Functions
lmc-ATPase •Group
Crude
Defined
activity
Interaction with ATP
WT G61D
++
++
++
+
H344D
--
,G429R
+
gpl9
--
DNA cleavage
Prohead binding
Motif
+
+
+ +
+ +f + + f
M-i
+
A-i
+
+ + +
+
N.I).
A-Ill
+
N.D.
+
N.I).
--
+
+
+
N.D.
G424E H347R
--
+ + + + + +
+ + + N.D.
+ + +
+
N.I). N.I). N.I).
A-I A-I I A-If
K64T K370I G429L K430T H553L
++ ++ + ++ ++
N.D. ++ +++ ++ N.I).
+ + + + +
+ + + + +
N.I). N.I). N.i). N.I). N.I).
G63D G367D G369D
+ + + + +
+ + + + +
A-III
M-I A-I A-If A-Ill A-Ill M-II
Data are summarized by qualitatively representing mutant activities relative to gpl9-wt activities as + + (DNA packaging and pewATPase) or + (others): packaging activity. Table 2: pac-ATPase activity. Table 3: interacti¢m with ATP. Figs 2. 6 and unpublished results: DNA cleavage, Figs 2, 3 and unpublished results: prohead binding. Fig. 6. and unpublished results. ATP and Mg2÷-binding motifs are abbreviated as A and M, respectively. N.D. not determined. t Inaet.ive binding.
(gp19-K64T, gp19-K370I, gp19-G429L, gp19K430T, gp19-H553L). Digestion p a t t e r n s of g p l 9 by several proteases were altered by the addition of ATP, indicating t h a t g p l 9 changes its conformation upon binding to A T P (Fujisawa et at., 1991). With the group 1 m u t a n t s , their trypsin digestion p a t t e r n s were different from t h a t of g p l 9 - w t in the absence of A T P and not significantly altered by the addition of A T P (Fig. 6). With gpl9-G61D, a b o u t 20 molecules bound to a prohead, independent of the presence or absence of ATP, unlike g p l 9 - w t for which six molecules bind the prohead in the presence of A T P and 20 bind in the absence of A T P (Fig. 4). The non-specific endonucleolytic activity of g p l 9 - G 6 1 D was suppressed by gpl8, like t h a t o f g p l 9 - w t , but not suppressed by A T P . u n l i k e t h a t of g p l 9 - w t ( K i m u r a & Fujisawa. 1991), indicating t h a t the gpl9--ATP interaction is lost in gpl9-G61D, gp19-G63D, another m u t a n t in the A T P binding domain-I, had decreased DNA packaging activity in the defined in vitro system. Gly61 and Gly63 are located in the A T P binding domain t h a t is conserved a m o n g the large subunits of packaging proteins of m a n y phages (Guo et al., 1987b; Table 1). These facts suggest t h a t A T P binding d o m a i n - I is involved in ATP-binding. With gp19-H344D, a b o u t 20 molecules bind to a prohead, independent of the presence or absence of ATP, but the ratios varied with experiments, suggesting t h a t the H344D m u t a t i o n m a y induce a conformational change in g p l 9 , resulting in unstable binding to a prohead. His 344 constitutes a potential Mg 2+ binding domain, His-X2-His (Berg, 1986; Guo et al., 1987b), which is conserved between T3 and T7 (Dunn & Studier, 1983; Y a m a d a et al., 1986). The
requirement of Mg 2+ for T3 DNA packaging has been d e m o n s t r a t e d (Fujisawa et al., 1978). Tile complete defect in DNA packaging activity of gpl 9H344D suggests t h a t this domain is involved in interaction with Mg 2+. g p l 9 - G 6 1 l ) - p r o h e a d or g p i 9 - H 3 4 4 D - p r o h e a d complexes were defective in DNA packaging. I t is reasonable t h a t these m u t a n t s lost the pac-ATPase activity because I)NA packaging is not initiated with these mutants. From the observations t h a t the tryptic digestion patterns of these m u t a n t s were different from t h a t of g p l 9 - w t , the possibility t h a t an amino acid sul)stitution causes a conformational change in the g p l 9 molecules, resulting in impaired activity, not by disruption of the predicted activity, A T P or Mg 2+ binding activity, cannot be excluded. The same a r g u m e n t would hold true for following discussions. Only one group 2 m u t a n t , gp19-G429R, had an increased pac-ATPase activity in spite of one-fifth i)NA packaging activity of g p l 9 - w t in both tile crude and defined systems ( T a b l e s 2 and 3). Therefore, gp19-G429R would require eight times more A T P for translocation of the same length of DNA than gpl9-wt. I t should be noted t h a t gp19-G429L, another substitution m u t a n t of Gly429, had an increased pac-ATPase activity (Table 3). These results suggest t h a t Gly429 in the A T P binding d o m a i n - I l l is i m p o r t a n t in coupling of A T P hydrolysis to I)NA translocation. With these mutants, although a lag period before the first appearance of m a t u r e heads was not different fi'om t h a t of gp 19-wt, it took a longer time tbr the conversion of most 5 0 S complexes to m a t u r e heads (Fig. 5), indicating t h a t the rate of DNA translocation slows down, on average, or t h a t it takes a
Mutant DNA
P a c k a g i n g Proteins of Phage T 3
much longer time for initiation of DNA translocation, starting from the precursor 50 S complexes. With gp19-G429R, the phage yields decreased more severely when started from the 50 S complex (Table 2 and Fig. 5), suggesting that the 50S complex containing gp19-G429R may be unstable and be inactivated during incubation with ATP-~-S. The group 3 mutants were active in DNA packaging in the defined system, but defective in the crude system. In the crude system, DNA is packaged by way of concatemer as an intermediate and monomeric I)NA must be cut from the concatemer (Fujisawa et al., 1978). The group 3 mutants except gp19-H347R were defective in both the non-specific endonuclease activity and the specific cleavage activity which is coupled to DNA packaging as gp19-G424E (Kimura & Fujisawa, 1991). The fact that these defects were produced by single amino acid substitutions indicate that the same domains of gpl 9 are responsible for the specific and non-specific cleavage activities. The group 3 mutations except the H347R mutation would lead to the defect in the con(.atemer processing, without affecting their ability to package mature T3 DNA. The mechanism which changes the non-specific cleavage activity to the specific cleavage activity necessary for" processing of concatemers remains to be elucidated. There are at least three activities required for the specific cleavage: cleavage sequence recognition, nucleotide binding and phosphodiester bond cleavage. Although cleavage of the concatemer junction was not observed, the longer fragment containing the terminally redundant sequence appeared when the reactions were treated with 1)Nasel (Fig. 3), indicating that the cleavage sequence, the left end of the terminally redundant sequence, is recognized and bound specifically, but not cleaved by these mutants. Since DNA cleavage activity is controlled through interaction with ATP, mutations in the ATP binding domains may induce a conformational change of gpl9 to inactivate the cleavage activity. Mutations in group3, except H347R, are scattered among the three ATP binding domains. These results suggest that these domains are close to each other in the gpl9 molecule to constitute a single functional domain or that several domains are involved in the endonucleolytic activity. The location of the endonuclease activity domains in the large subunit of the packaging proteins may be general (T4, Rao & Black, 1988: 2, l)avidson & Gold, 1992). In ),, mutations in two distinct domains of the large subunit gpA inactivate the endonuclease activity of the terminase, without disrupting the DNA packaging activity (Davidson & Gold. 1992). In addition to gpA, the cleavage at cos sequences depends on the small subunit gpNul, which is involved in recognition and binding of the co.~ sequence (Frackman et al., 1985). Mutations in or near the ATP binding motif of gpNul alter the interaction of terminase with the cos sequence (Feiss et al., 1988; Cue & Feiss, 1992). In spite of being a group 3 mutant, gpl9-H347R
257
was active in both endonuclease activities. Since the specific endonuclease activity in the defined system corresponds to the termination cleavage of the concatemer processing (Fujisawa et al., 1990), this mutant would be defective in the initiation events. We have not characterized the group 4 mutants ill detail because these mutants were active in DNA packaging in both the defined and crude systems. Although the importance of Lys to ATP binding domains is clear (Fry et al., 1986; Maruyama & MacLennan, 1988; Rosen et al., 1989; Xia & Storm, 1990; Hinz & Kirley, 1988), Lys in three ATP binding domains may not be crucial for interaction with ATP (Table 2), as the case for the transcription termination factor, rho (Dombroski et al., 1988). As discussed in a previous paper (Kimura & Fujisawa, 1991), His553 may not be involved in Mg~+ binding because His553 is not conserved between T3 and T7, although three ATP binding motifs and another Mg2+ binding motif are conserved (Dunn & Studier, 1983; Yamada et al., 1986). As shown in the present paper, gpl9 has multiple ATP and Mgz+ binding motifs in its amino acid sequence, and mutations in these motifs of gpl9 molecules resulted in different defects in DNA packaging process: abortive interactions with ATP, abnormal binding to prohead, loss of the nonspecific and specific endonuclease activities and uncoupling of ATP hydrolysis to DNA translocation. These facts indicate that gpl9 plays crucial roles in DNA translocation and in processing of concatemer DNA. Since the same defects were observed with mutants in different motifs, multiple domains would be implicated in each function. The analyses of the DNA packaging process in the defined systems of T3 and ~b-29 demonstrate that the large subunits, gpl9 ofT3 (Shibata et al., 1987a; Fujisawa et al., 1991) and gpl6 of ~-29 (Guo et al., 1987a), undergo conformation changes in multiple steps of the DNA packaging process through interaction with ATP. In T3, six molecules of gpl9 bind to the connector at the portal vertex to form the functional packaging machinery in the presence of ATP. When gpl9 is bound to proheads, the gpl 9-prohead complex gains the ability to bind to a gpl8-DNA complex in the presence of ATP. ATP binding and hydrolysis in the prohead/gpl9/gpl8/ DNA complex result in DNA translocation into the head. Although speculative models for DNA translocation have been proposed, much attention has not been given to the role of the terminase or packaging enzyme in these models (Earnshaw & Casjens, 1980; Turnquist et al., 1992). In a previous paper (Shibata et al., 1987b), we proposed a ratchet model for DNA translocation in which the gpl9-connector complex located at the portal vertex would translocate DNA into the prohead by a cyclic change in the conformation of gpl9 mediated by ATP. In the model, one molecule of gpl9 is bound to each component of six domains of the connector and one of the gpl9 molecules is bound to the sugar-phosphate backbone of duplex DNA. When ATP is hydrolyzed, gpl9 in the
258
M u t a n t D N A Packaging Protein8 of Phage T 3
compiex contracts, forcing the D N A into the head, and then dissociates from the sBgar-phosphate backbone. A t this m o m e n t , the n e x t g p l 9 binds the neighboring s u g a r - p h o s p h a t e backbone. The same cyclic changes translocate D N A into the head. Ordered conformational changes in proteins t h a t are -driven b y nucleotide t r i p h o s p h a t e hydrolysis provide explanations for A T P - d r i v e n biological machines (Alberts & Miake-Lys, 1992). Rao & Black (1988) proposed a p a c k a s o m e model in which gpl7, the large subt~nit of T4 terminase, constitutes the packaging machinery. According to the model, D N A is translocated into the prohead by a complex structure assembled a t the portal vertex of the prohead utilizing A T P hydrolysis energy. Much remains to be learnt a b o u t the functions of the packaging enzymes or terminases in DNA packaging. We are grateful to Dr Michael Feiss (The University of Iowa) for his invaluable help with the manuscript. This work is supported by a grant-in-aid for scientific research from the Ministry of Education of Japan.
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Edited by N. Sternberg (Received 19 A p r i l 1993; accepted 24 August 1993)