- Email: [email protected]
[16] pGT5 replication initiator protein Rep75 from Pyrococcus abyssi
[1 6]
Rep75 FROM P y r o c o c c u s a b y s s i
193
[16] pGT5 Replication Initiator Protein Rep75 from Pyrococcus abyssi By STI~PHANIEMARSIN and PATRICK FORTERRE
Introduction Plasmids are extrachromosomal elements that can replicate autonomously in host cells. They are well known and studied in bacteria and have allowed the development of essential genetic tools in many bacterial species. Plasmids have also been very useful in studying fundamental cellular mechanisms, such as DNA replication. The plasmid pGT5 (3.4 kb), isolated from the euryarchaeon P y r o c o c c u s abyssi, was the first plasmid studied in hyperthermophilic archaea.1 It was completely sequenced and shown to replicate via the rolling circle (RC) mechanism (see below). This asymmetrical replication mechanism, which was first described for q~X174 and m13 coliphages, 2 is also used by many bacterial plasmids and eukaryotic viruses. 3-8 RC replicons encode a replication initiator protein (usually named Rep) that exhibits a site-specific endonuclease/ligase activity. Rep proteins recognize the plasmid double-stranded origin (dso) and cleave the plus strand, leaving a free Y-hydroxyl end that will be used as primer for DNA chain elongation by the host replicase. During the replication round, Rep proteins usually remain covalently linked to the 5' end of the displaced DNA chain. At the end of one replication round, Rep proteins cleave the d s o again and religate the new synthesized strand, generating a circular single-stranded form that is converted into a double-stranded plasmid by host proteins. Several Rep proteins encoded by bacterial or eukaryal RC replicons have been studied in vivo and in vitro, and they all share common characteristics. 9-14 In vitro, 1 G. Erauso, S. Marsin,N. Benbouzid-Rollet,M. E Baucher, T. Barbeyron,Y. Zivanovic,D. Prieur, and P. Forterre, J. Bacteriol. 178, 3232 (1996). 2 A. Kombergand T. Baker, "DNA Replication,"2nd ed. W. H. Freeman& Co. 1992. 3 G. del Solar, R. Giraldo,M. J. Ruiz-Echevarria,M. Espinosa,and R. Diaz Orejas, Microbiol. Mol. Biol. Rev. 62, 434 (1998). 4 M. Espinosa,G. del Solar, E Rojo, and J. C. Alonso,FEMS Microbiol. Letr 130, 111 (1995). 5 A. Gruss and S. D. Ehrlich,Microbiol. Rev. 53, 231 (1989). 6 S. A. Khan,Microbiol. Mol. Biol. Rev. 61, 442 (1997). 7 E. V. Kooninand T. V. Ilynia,J. Gen. Virol. 73, 2763 (1992) 8 R. P. Novick,Ann. Rev. MicrobioL 43, 537 (1989). 9 R. Hanai and J. C. Wang,J. Biol. Chem. 268, 23830 (1993). l0 M. E Noirot-Gros,V. Bidnenko,and S. D. Ehrlich,EMBO J. 13, 4412 (1994). I1 j. Laufs, S. Schumacher,N. Geisler,I. Jupin, and B. Gronenborn,FEBSLett. 377, 258 (1995). 12 C. D. Thomas, D. E Balgon,and W. V. Shaw,J. Biol. Chem. 265, 5519 (1990). 13 M. Moscoso, R. Eritja, and M. Espinosa,J. Mol. Biol. 268, 840 (1997). 14R. A. Hoogstraten,S. E Hanson,and D. P. Maxwell,MPMI 9, 594 (1996).
METHODS1NENZYMOLOGY,VOL.334
Copyright©2001byAcademicPress Allrightsofreproductioninanyformreserved. 0076-6879/00$35.00
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NUCLEIC ACID MODIFYING ENZYMES
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A
Orf2 I (ll91bp)[
~kgtg I
P_G T5
(3444bp) /Orfl (1965bp)
B 1
85
219
I
I
I
I
I
(atg)
(gtg)
(gtg) I
motifs II 111 •
I
Rep50 Rep60 Rep75
654 I
1
i
FIG. 1. pGT5 organization. (A) Map of pGT5. The two major ORFs ( 1 and 2) and their orientation are indicated by thick arrows. The three possible initiation codons for Orfl and the putative dso and sso are noted. (B) Map of Rep75 encoded by ORF1. The three motifs (1-3) common to Rep proteins from the ~ X 174/pC 194 superfamily of RC replicons are indicated by black boxes. The positions of the three possible initiation codons are indicated.
they can cleave and religate an oligonucleotide harboring the dso sequence; the cleavage is always done by a transesterification with a tyrosine at the active site. Several superfamilies of RC replicons have been identified, based on sequence similarities among their Rep proteins.15 One of them, the dpX 174/pC 194 superfamily, includes coliphages, bacterial plasmids, and eukaryal geminiviruses. In particular, the plasmid pRQ716 from the hyperthermophilic bacterium Thermotoga sp. RQ7 and pGRB 17 from the archaeon Halobacterium sp. GRB belong to this superfamily. Rep proteins of the qbX 174/pC 194 superfamily are characterized by three conserved motifs, 1, 2, and 3, which are present in the same arrangement from the N- to C-terminal.~5 The motif 3 contains either one or two active tyrosines involved in the cleavage and reactions of transphosphorylation. The two putative proteins encoded by pGT5 (orfl and orf2, Fig. 1A) have no detectable similarity 15 T. V. Ilyina and E. V. Koonin, Nucleic Acids Res. 20, 3279 (1992). 16 j. S. Yu, and K. M. Noll, J. Bacteriol. 179,7161 (1997). J7 N. R. Hackett, M. P. Krebs, S. DasSarma, W. Goebel, U. L. RajBhandary, and H. G. Khorana, Nucleic Acids Res. 18, 3408 (1990).
[ 16]
Rep75
FROM
Pyrococcus abyssi
195
with other proteins in databases using search programs such as FASTA or BLAST. However, the three motifs characteristic of the q~X174/pC194 Rep superfamily can be detected by visual inspection in the putative protein of 75 kDa encoded by orfl.1 Moreover, an 11 nucleotide sequence, identical to the pC 194 dso, is present in the pGT5 sequenceJ These two observations suggested that pGT5 replicated via the RC mechanism and, as a result, could be another archaeal member of the qbX 174/pC 194 superfamily.15 This prediction has been confirmed by biochemical analyses. It was shown that: (1) cells ofP. abyssi contain a single-stranded form of pGT5 that has the characteristic of a RC replication intermediate I; (2) the purified Rep protein of pGT5 (Rep75) exhibits a highly thermophilic and specific nickingclosing (NC) activity on single-stranded oligonucleotides containing the pGT5 dso sequence 18; and (3) replacement of the only tyrosine located in motif 3 by a phenylalanine abolishes this activity.19 In addition to its expected activities (NC), Rep75 exhibits an unusual site-specific nucleotidyl-terminal transferase (NTT) activity, never described before for a Rep protein. 18This transfer occurs only at the 3' end of the nicking site with an adenine (or a deoxyadenine) nucleotide monophosphate, and it was proposed that this NTT activity plays a role in the regulation of pGT5 replication. 19 The protein Rep75 can be overproduced in Escherichia coli and purified to near homogeneity. The method used, as well as the activity tests, is described in detail here and could be relevant to the study of other hyperthermophilic Rep proteins. It is still not known if Rep75 corresponds to the physiological form of the pGT5 Rep protein., since the orfl of pGT5 contains three in-frame initiation codons (Fig. 1), and thus could encode for three putatives Rep proteins of 75, 66, and 50 kDa, respectively (Rep75, Rep60, and Rep50). In particular, since all Rep proteins from bacterial plasmids and viruses, as well as Rep proteins from geminiviruses, are smaller than Rep75 (in the range of 20 to 40 kDa), the physiological form of pGT5 Rep might have been Rep50 or Rep60. This chapter also describes the purification of the Rep50 protein expressed in E. coli and its biochemical characterization. Results have shown that Rep50 cannot be the physiological form of pGT5 Rep and provide novel information about the mechanism of Rep75 activity. Unfortunately, the rep60 gene has yet to be cloned in E. coli, because of the instability of this DNA fi'agment in pET3b vector. P u r i f i c a t i o n of R e c o m b i n a n t p G T 5 R e p 7 5 P r o t e i n
Cloning of rep75 Gene The Rep proteins of RC plasmids are usually present in very low copy number in the host cell. In bacteria, these proteins have been successfully purified from 18S. Marsin and E Forterre,Mol. Microbiol.27, 1183(1998). 19S. Marsin and P. Forterre,Mol. MicrobioL33, 537 (1999).
196
NUCLEICACIDMODIFYINGENZYMES
[1 6]
host cells only when it has been possible to take advantage of mutations that upregulate Rep expression in vivo. In the absence of such system, Rep proteins should be overproduced using expression vectors. The bacteriophage T7 expression system (pET3b plasmid from Stratagene, La Jolla, CA) is very efficient to obtain high amount of the pGT5 Rep75 proteins in E. coli. In this plasmid, the gene of interest is under the control of the bacteriophage T7qbl0 promoter and located downstream of a strong ribosome binding site. T7 RNA polymerase is provided by isopropylthiogalactoside (IPTG) induction of the T7 RNA polymerase gene under control of the lac repressor in a defective integrated lambda bacteriophage. The rep75 gene has been cloned into the pET3b plasmid from PCR products amplified from the pGT5 plasmid using Vent polymerase ~8 (New England Biolabs, MA). The recombinant plasmid is called pETRep75.
Cell Culture and Induction
Cells of E. coli BL21(hDE3) pLysS, carrying the plasmid pETRep75, are grown at 37 ° in L-broth containing 100 Ixg/ml ampicillin and 25 Ixg/ml chloramphenicol with vigorous aeration up to an absorbance of 0.6 at 600 n m (A600). The Rep75 protein is induced by addition of 0.5 mM IPTG followed by growth for 3 or 4 hr up to an A600 close to 1. After induction, Rep75 can represent up to 30% of E. coli proteins (compare TO and T4 on Fig. 2). The cells are then harvested by centrifugation without previous chilling, to prevent lysis, and kept frozen at - 2 0 ° .
(kDa) M 205 116 66 55 45 36
TO
T4
S
Wl
W2
IB
PC
29 24 19 FIG.2. SDS-PAGEanalysisof Rep75 purification. The proteins were analyzedby electrophoresis usinga 10%gel stainedwithCoomassieBrilliantBlueandphotographed.M, molecularsizemarker;TO, E. coli crude extract of uninducedculture; T4, E. coli crude extract after4 hr of induction; S, soluble proteins after sonication;W1 and W2, first and second wash, respectively,in buffer C of insoluble proteins; IB, aggregatedRep75resolubilizedin bufferD; PC, Rep75elutionfromthe phosphocellulose column.
[ 16]
Rep75 FROM Pyrococcus abyssi
197
Purification of lnclusion Bodies Cells from 100 ml culture are resuspended at an A60o of 30 in 3 ml of ice-cold buffer A300 (25 mM Tris-HC1, pH 8.8, 300 mM NaC1, 2 mM dithiothreitol, and 1 mM EDTA) and broken by sonication. Three ml of ice-cold TE buffer is then added, and the viscosity due to DNA is reduced by incubation with 10 txg/ml DNase I in the presence of 8 mM MgC12 for 1 hr at 4 °. Three ml of ice-cold buffer B [ 10 mM Tris-HC1, pH 8.8, 100 mM NaC1, 1% Nonidet P-40 (NP-40), 0.5% deoxycholate, and 1% Triton X-100) is added and mixed to solubilize membranes. Inclusion bodies containing Rep75 are collected by centrifugation at 10,000g for 10 min. The supernatant contains very little detectable soluble Rep75 protein (compare fractions T4 and S in Fig. 2). The pellet is washed twice in ice-cold buffer C (50 mM Tris-HC1, pH 8.8, 500 mM NaC1, and 1 mM EDTA). Part of Rep75 is lost in the first wash (W1), but this step eliminates some contaminants (compare fractions W1 and IB, Fig. 2). The insoluble material present in the pellet is solubilized in 1 ml of buffer D (10 mM Tris-HC1, pH 8.8, and 6 M guanidinium hydrochloride) (fraction IB, Fig. 2). Rep75 is completely solubilized by this procedure, whereas it is only partly solubilized in 8 M urea.
Renaturation and Purification of Solubilized Rep 75 Fraction IB is diluted in buffer D down to a protein concentration of 0.1 mg/ml. Solubilized proteins present are renaturated by continuous dialysis overnight at 4 ° against buffer A200 (buffer A300 with 200 mM NaC1 instead of 300 mM). A large amount of protein precipitates during this step (see comments) and the aggregate is eliminated by centrifugation for 10 min at 15,000g. The supernatant is loaded on a 2 ml P11 cellulose phosphate (Whatman, Clifton, NJ) column equilibrated in buffer A200 and then washed with five volumes of buffer A300. Rep75 is eluted with 5 ml of buffer A450 (buffer A300 with 450 mM NaC1 instead of 300 mM) (fraction PC, Fig. 2). Rep75 can be stored in this buffer with 0.08% sodium azide for 1 month at 4 °, and for at least 1 year at - 8 0 ° without loss of activity.
Comments about Rep75 Purification Soluble recombinant Rep75 protein could not be obtained by changing the conditions of induction, such as growth temperature or IPTG concentration. A purification procedure was therefore designed that takes advantage of Rep75 insolubility, using the isolation of inclusion bodies as an enrichment step. There were significant amounts of Rep75 in inclusion bodies, corresponding to about 5 mg per 100 ml of culture. Rep75 was recovered after solubilization of the inclusion bodies followed by renaturation of the protein. During the renaturation step, 70 to 80% of the protein aggregated. This percentage increases if the initial Rep75
198
NUCLEIC ACID MODIFYINGENZYMES
[16]
concentration is more than 0.1 mg/ml. Aggregation also occurs in the phosphocellulose column if the column volume is less than 1 ml for 1 mg of soluble protein. Moreover, after elution, less than 0.25 mg/ml of Rep75 protein was obtained, and it was not possible to concentrate Rep75 further. Nevertheless, between 0.5 mg and 1 mg of pure recombinant Rep75 could be obtained routinely per 100 ml of culture. Rep75 is >95% homogeneous as determined by Coomassie Blue stained SDS-PAGE. Rep75 preparations obtained after denaturation/renaturation could a priori contain both inactive (incorrectly folded) and active proteins. This is likely, in view of the amount of protein required to get in vitro activities (see below). It is not possible to perform quantitative enzymatic analysis with recombinant Rep75 since it was not possible to determine the proportion of active protein in the final fraction (PC, Fig. 2). Preparations sometimes contained low amount of polypeptides that have molecular weights, slightly lower than those of Rep75 and that cross-react with Rep75 antibodies (data not shown). These proteolytic products of Rep75 are as thermostable as full-size Rep75 protein (Fig. 3A). Figure 3A shows that only 60% of recombinant Rep75 remained soluble when the purified protein is incubated for 1 hr at 100 °. However, the protein preparation retains activity and its level remains constant (this is the case, for example, for the nicking activity illustrated in Fig. 3B). This suggests that the polypeptides that disappear during incubation at 100 ° are those that have not been correctly refolded during the renaturation step of the purification procedure. One can thus conclude that correctly folded and active Rep75 proteins are fully stable for at least 1 hour at 100 °.
A Incubation time at 100° 0 5 15 30 60 !
T
B Preincubationtime ofRep75at100°(rain) 0 5 10 15 30 60
i i
*I6L-9R
Rep75~
*16L
FIG.3. Rep75 thermostability. (A) Rep75 remains soluble after incubationat 100°. Purified Rep75 was incubated at 100° during the indicated time and centrifuged for 10 min at 15,000g to eliminate insoluble material. Soluble Rep75 proteins were then loaded on a 10% SDS-PAGE.The gel shown was stained with Coomassie Brillant Blue. (B) Rep75 remains active after incubation at 100°. The Rep75 protein incubated at 100° during the indicated time was used in the nicking test. The labeled oligonucleotide substrate *16R-9Land its cleavage product *16L are indicated.
Rep75 FROM Pyrococcus abyssi
[ 16]
199
Recombinant pGT5 Rep50 Purification The rep50 gene has been cloned in the pET3 plasmid, using the same strategy as for the rep75 gene. However, in contrast to the plasmid pETRep75, the plasmid harboring the rep50 gene (pETRep50) is unstable in the BL2 IpLysS strain and is lost during the culture. To express Rep50, several colonies of transformed cells are thus selected on plates and used immediately as inoculum for the overproducing culture. Rep50 was then expressed, as described for Rep75. The protein Rep50 is also insoluble and can be recovered from purified inclusion bodies. Rep50 is solubilized in I ml of buffer D and renatured simply by a 10-fold dilution in buffer A300. Some aggregated material is then removed by centrifugation for I0 min at 15,000g. In contrast to Rep75, Rep50 does not bind to a phosphocellulose column; however, the protein Rep50 is at least 90% pure (similar to the degree of Rep75 purity in fraction IB). Approximately 0.5 mg of Rep50 protein is obtained from 100 ml of culture.
Assay Method The activity of Rep proteins from RC plamids can be assayed on the basis of their ability to support in vitro replication initiation of RC plasmids. This is very tedious since it requires one to set up a complete in vitro system with host proteins. The basic function of Rep proteins is nicking-closing activity, which is usually specific for DNA-containing the dso plasmid sequence. This activity can be detected on supercoiled plasmid as a topoisomerase activity, or on single-stranded oligonucleotides containing the dso nicking site. The latter facilitates better analysis of the reaction, since it is possible to work with small oligonucleotides (less than 50 nucleotides) of defined sequence and to test either nicking or closing activity, as well as the novel terminal transferase activity discovered in Rep75. In tests for the nicking-closing activity of Rep75 and Rep50, it was noted that oligonucleotides as short as 15-25 nucleotides could be used. The oligonucleotides substrates
TABLE I OLIGONUCLEOTIDES USED IN ACTIVITY TESTSa Name
Sequence
Size
16L-9R 9L- 16R 16L
51-CGTTGGGTTTATCTTG/ATATATCCA-3 ~
25 nt 25 nt 16 nt
5'-TTTATCTTG/ATATATCCACAACCAA-3 ~ 51-CGTTGGGTTTATCTTG
a The characters in boldface type indicate the conserved nucleotides between the putative dso pGT5 sequence and the dso sequence from pC194. The slash indicates the cleavage site.
200
NUCLEIC ACID MODIFYING ENZYMES
[ 16]
routinely used are presented in Table I. They are radiolabelled at their 5' end using [~_32 P]ATR To study the affinity of Rep75 or Rep50 proteins to DNA, the gel shift technique was used, which turned out to be suitable for following binding of the protein on both single-stranded or double-stranded oligonucleotides.
Nicking-Closing For nicking-closing reactions, 20 fmol of the indicated oligonucleotide is incubated at 105 ° with Rep75 in 10 txl of buffer R (50 mM HEPES-HC1, pH 8,200 mM Sodium glutamate, 1 mM DTT, 5 mM MnCI2, 0.1% Triton X-100, 1 mM EDTA, and 50 txg/ml BSA). For ligation tests, the incubation is performed in two steps, first 5 min at 105 ° and then 5 min at 75 °, because of the different temperature requirement between nicking and closing activities (see below). Reactions are stopped on ice by addition of EDTA 25 mM and incubation with 15 txg of proteinase K for 30 min at 55 °. Five ~1 of loading buffer is then added (50 mM Tris-borate, pH 8.3, 80% deionized formamide, 1 mMEDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). The reaction products are separated on a 20% polyacrylamide (20 : 1) gel containing 8 M urea. Autoradiograms are performed and quantified by scanning the gel with a Phosphoimager ImageQuant.
Nucleotide Terminal Transferase 16L oligonucleotide (Table I), harboring only the left part of the nicking site, is radiolabeled and used in NTT tests. The assay is performed at 75 ° in the same buffer as described for the NC test but with 1 mM of ATP or dATE
Gel Shift Twenty fmol of 5'-radiolabeled single-stranded or double-stranded oligonucleotides is incubated with 1.3 pmol of the Rep protein (protein to DNA molar ratio of 60 : 1) for 5 min. The reaction is stopped by addition of 25 mM EDTA and 5 txl of loading buffer (30% glycerol, 0.25% xylene cyanol, and 0.25% bromphenol blue). The products are then loaded on a 8% polyacrylamide gel (30: 1) in 5% glycerol, 0.25× TBE, and run in 0.25× TBE at room temperature. Incubation temperature is not important for migration under native conditions, and no differences are observed when protein and DNA are previously incubated together either at 105 ° and 4 ° . C o m m e n t s a b o u t R e p 7 5 Activities
Rep75 Nicking-Closing Activity Figure 4 illustrates Rep75 nicking-closing activities on single-stranded oligonucleotides harboring the cleavage sequence of pGT5 dso. The 5' radiolabeled *16L-9R is cleaved by Rep75, so that only the *16L nicking product can be
Rep75 FROM Pyrococcus abyssi
[16]
Reaction type
Nicking I
201
Nleking/Closln 8 NTT I I
II
I
*16I_,-16R *9L-16R
*16L-A *I6L
*9L FIG.4. In vitroRep75 activities. Results of nicking, nicking--closing,and NTT tests were run on a polyacrylamide gel and autoradiographed. The radiolabeled oligonucleotides,used as substrates, are indicated on the upper part. The oligonucleotidesdetectable on the gel are indicated on the sides of the figure. M, markers. detected. The same activity is obtained with the *9L-16R oligonucleotide, which is cleaved to give the *9L product. When the two substrates *16L-9R and *9L16R are incubated together with Rep75, the two nicking products are obtained and the closing product *16L-16R can be detected (the ligation efficiency is too weak here to observe the *9L-9R closing product). A *16L-16R religation product is also obtained when *9L-16R is incubated with 16L and Rep75. No religation is observed when the test is performed with oligonucleotides containing only the right or the left part (16L and 16R oligonucleotides) of the nicking site (data not shown). This is because Rep75 should activate the DNA by nicking and covalent fixation before the religation step at the 3' end. TM
Rep 75 NTT Activity In the presence of ATP or dATE Rep75 exhibits an unusual site-specific nucleotidyl transferase activity, i.e., it transfers one AMP or dAMP to the 3'OH extremity of an oligonucleotide containing the left part of the nicking site. Figure4 illustrates an experiment in which the *16L oligonucleotide (Table II) is transformed into a 17-mer long oligonucleotide (* 16L-A) by this activity. The
202
NUCLEIC ACID MODIFYINGENZYMES
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T A B L E II ACTIVITY COMPARISON BETWEEN REP75 AND REP50 a D N A binding
Source Protein Rep75 Rep50
Activities Nicking + +
ssDNA
Closing + .
NTT
Left part
Rightpart
Left pan
Rightpan
+
+
nd
+ nd
+ .
.
dsDNA
.
The activityassayswere performedunder the same conditionswith the two proteins. DNAbinding was performed by gel shift assay as described in the text, on oligonucleotidesingle-stranded (ss) DNA or double-stranded (ds) DNA, these substrates containing the left part or the right part of the nicking site. nd, Not determined.
transfer is slightly better with dATP than with ATP as substrate (Fig. 5C), and the activity is already maximal at 50 R~M. The NTT activity is nucleotide specific, since Rep75 cannot use (d)CTP, (d)GTP, (d)TTP, AMP, ADP, or AMP-PNP. The NTT protein active site at least partly overlaps the NC active site, as an arginine located in the motif 3 of Rep75 is essential for both ligation and NTT activities. However, the nicking and NTT activities can be uncoupled in vitro, since the active tyrosine in motif 3 is dispensable for the NTT activity, and the arginine of motif 3 is dispensable for the nicking activityJ 9 The NC and NTT activities of Rep75 reach equilibrium after 15 min of incubation. Optimal results are obtained when Rep75 is added at a very high protein to DNA molar ratio (60 : 1), Is indicating the low activity of the protein preparation. However, this is not unusual for this type of protein. In nicking assays, one never observes more than 50% of cleavage for the conditions studied, whereas it is possible to observe nearly complete transfer in NTT assays. Moreover, it is difficult to obtain the same specific activity for different protein preparations, probably due to the denaturation step in the purification process.
Comparison of Different Activities of Rep75 The different Rep75 activities are optimal at different assay conditions. The most important and surprising difference is their temperature optima. Figure 5A shows that NTT activity is optimal at 75 °, whereas the nicking activity increases with temperature at least up to 105 ° (verified in a test tube using a thermoprobe). The closing activity is also known to be optimal around 85 ° . 19 The reason for these differences is presently unclear. Another important observation is the difference in the divalent cation requirement between nicking and NTT activities (Fig. 5B). The two activities are optimal
[16]
Rep75
FROM
Pyrococcus abyssi
203
A actlvit', %
activity % 100
60'
transfer % "
75-
80 • 60-
40.
40. 25"
20 20"
0~ 35 45 55 65 75 85 95 105 Incubation temperature
0
2,5
5 7,5 mM cation
10
,
,
//
0 50 100
1000 nucleotide
FIG. 5. Different characteristics of Rep75 activities. (A) Temperature dependence of Rep75 activities. Percentage activity corresponds to the percentage of modificated oligonucleotides, appearance of cleavage product in the case of nicking test (black squares), and apparition of transfer product in the case of NTT assay (white squares). (B) Effect of variation in Mg 2+ and Mn 2+ concentration on Nicking and NTT activities. The activities assays were performed in presence of Mn 2+ (circles) or Mg 2+ (squares). NTT activity (in black) and nicking activity (in white) were quantified as presented in A. (C) Effect of variation in ATP and dATP concentration on NTT activity. The NTT assays were performed for different concentrations of ATP (open circles) or dATP (black circles).
for 5 mM of MnC12. However, the NTT activity is more specific for this salt, since only 25% of the nicking activity is conserved when MnC12 is replaced by MgC12. On the other hand, the NTT activity cannot be observed at all in the presence of MgC12.
Comparison between Rep75 and Rep50 Partially purified Rep50 and Rep75 were compared (Table II). This makes sense since partially purified Rep75 (fraction IB, Fig. 2) exhibits the same activities as the completely pure protein (fraction S). Rep50 can perform site-specific DNA cleavage and its nicking activity is optimal at 105 °, as in the case of Rep75. However, unlike Rep75, Rep50 does not have closing or NTT activities. This clearly indicates that Rep50 cannot be the native protein produced by pGT5 in vivo. The disappearance of the ligation and NTT activities in Rep50 can be explained by the inability of this truncated protein to recognize single-stranded DNA. Indeed, unlike Rep75] 8 Rep50 cannot bind single-stranded DNA fragment harboring the left part of the nicking site, as indicated by gel retardation experiment (Table II). Rep50 could have lost these properties because the single-stranded DNA binding site of Rep75 is located in its N-terminal region, which has been removed from Rep50, or because the absence of this region induces an incorrect protein folding. This suggests that the nicking activity (conserved in Rep50) does not involve the
204
nicking site
5'
[16]
NUCLEIC ACID MODIFYING ENZYMES
Ot ~
+Rep75
O
ct 5'
Y
non covalent fixation
m,,._ 5'
local denaturation
3' 5'
| ~ nicking, covalent fixation
O
3'
~: ~i~i¸ .~a closing
5'
non covalent
interaction FIG. 6. Model of Rep75 origin recognition, a is the left part of the nicking site, 13 is the right part of the nicking site. The model is explained in the text.
strong interaction detected by gel shift between single-stranded DNA and Rep75. In contrast, this interaction should be essential for the closing and NTT activities. Comparison between Rep75 and Rep50 suggests a mechanism for origin recognition by Rep75 (Fig. 6). Rep75 first interacts with the right part of the nicking site (double-stranded) and induces the local melting of DNA. This is similar to the model for recognition of ~X174 dso by gpA, which has been proposed by Baas and Jans. 2° The single-stranded DNA region thus produced is then cleaved by Rep75, which remains covalently linked to the 5' end. The covalently linked protein could then interact with the left part (still single-stranded) of the nicking site and religates DNA. This last step cannot be performed by Rep50, since it is unable to interact with the left part of the nicking site. The proteins Rep75 and Rep50 appear to be an interesting model system to study the mechanism of action of RC Rep proteins, in general. Overproduction of these proteins could pave the way for structural studies on an exciting new family of enzymes that can perform different types of DNA manipulations (cleavage, ligation, and terminal transfer) using a single active site. Acknowledgments This work was supported by grants from the Association pour la Recherche contre le Cancer and the European Community Programm Cell Factory (BIO4-CT 96-0488).
20 E D. Baas and H. S. Jansz, Curr. Top. Microbiol. lmmunol. 136, 31 (1988).
3'
Rep75 FROM P y r o c o c c u s a b y s s i
193
[16] pGT5 Replication Initiator Protein Rep75 from Pyrococcus abyssi By STI~PHANIEMARSIN and PATRICK FORTERRE
Introduction Plasmids are extrachromosomal elements that can replicate autonomously in host cells. They are well known and studied in bacteria and have allowed the development of essential genetic tools in many bacterial species. Plasmids have also been very useful in studying fundamental cellular mechanisms, such as DNA replication. The plasmid pGT5 (3.4 kb), isolated from the euryarchaeon P y r o c o c c u s abyssi, was the first plasmid studied in hyperthermophilic archaea.1 It was completely sequenced and shown to replicate via the rolling circle (RC) mechanism (see below). This asymmetrical replication mechanism, which was first described for q~X174 and m13 coliphages, 2 is also used by many bacterial plasmids and eukaryotic viruses. 3-8 RC replicons encode a replication initiator protein (usually named Rep) that exhibits a site-specific endonuclease/ligase activity. Rep proteins recognize the plasmid double-stranded origin (dso) and cleave the plus strand, leaving a free Y-hydroxyl end that will be used as primer for DNA chain elongation by the host replicase. During the replication round, Rep proteins usually remain covalently linked to the 5' end of the displaced DNA chain. At the end of one replication round, Rep proteins cleave the d s o again and religate the new synthesized strand, generating a circular single-stranded form that is converted into a double-stranded plasmid by host proteins. Several Rep proteins encoded by bacterial or eukaryal RC replicons have been studied in vivo and in vitro, and they all share common characteristics. 9-14 In vitro, 1 G. Erauso, S. Marsin,N. Benbouzid-Rollet,M. E Baucher, T. Barbeyron,Y. Zivanovic,D. Prieur, and P. Forterre, J. Bacteriol. 178, 3232 (1996). 2 A. Kombergand T. Baker, "DNA Replication,"2nd ed. W. H. Freeman& Co. 1992. 3 G. del Solar, R. Giraldo,M. J. Ruiz-Echevarria,M. Espinosa,and R. Diaz Orejas, Microbiol. Mol. Biol. Rev. 62, 434 (1998). 4 M. Espinosa,G. del Solar, E Rojo, and J. C. Alonso,FEMS Microbiol. Letr 130, 111 (1995). 5 A. Gruss and S. D. Ehrlich,Microbiol. Rev. 53, 231 (1989). 6 S. A. Khan,Microbiol. Mol. Biol. Rev. 61, 442 (1997). 7 E. V. Kooninand T. V. Ilynia,J. Gen. Virol. 73, 2763 (1992) 8 R. P. Novick,Ann. Rev. MicrobioL 43, 537 (1989). 9 R. Hanai and J. C. Wang,J. Biol. Chem. 268, 23830 (1993). l0 M. E Noirot-Gros,V. Bidnenko,and S. D. Ehrlich,EMBO J. 13, 4412 (1994). I1 j. Laufs, S. Schumacher,N. Geisler,I. Jupin, and B. Gronenborn,FEBSLett. 377, 258 (1995). 12 C. D. Thomas, D. E Balgon,and W. V. Shaw,J. Biol. Chem. 265, 5519 (1990). 13 M. Moscoso, R. Eritja, and M. Espinosa,J. Mol. Biol. 268, 840 (1997). 14R. A. Hoogstraten,S. E Hanson,and D. P. Maxwell,MPMI 9, 594 (1996).
METHODS1NENZYMOLOGY,VOL.334
Copyright©2001byAcademicPress Allrightsofreproductioninanyformreserved. 0076-6879/00$35.00
194
NUCLEIC ACID MODIFYING ENZYMES
[ 16]
A
Orf2 I (ll91bp)[
~kgtg I
P_G T5
(3444bp) /Orfl (1965bp)
B 1
85
219
I
I
I
I
I
(atg)
(gtg)
(gtg) I
motifs II 111 •
I
Rep50 Rep60 Rep75
654 I
1
i
FIG. 1. pGT5 organization. (A) Map of pGT5. The two major ORFs ( 1 and 2) and their orientation are indicated by thick arrows. The three possible initiation codons for Orfl and the putative dso and sso are noted. (B) Map of Rep75 encoded by ORF1. The three motifs (1-3) common to Rep proteins from the ~ X 174/pC 194 superfamily of RC replicons are indicated by black boxes. The positions of the three possible initiation codons are indicated.
they can cleave and religate an oligonucleotide harboring the dso sequence; the cleavage is always done by a transesterification with a tyrosine at the active site. Several superfamilies of RC replicons have been identified, based on sequence similarities among their Rep proteins.15 One of them, the dpX 174/pC 194 superfamily, includes coliphages, bacterial plasmids, and eukaryal geminiviruses. In particular, the plasmid pRQ716 from the hyperthermophilic bacterium Thermotoga sp. RQ7 and pGRB 17 from the archaeon Halobacterium sp. GRB belong to this superfamily. Rep proteins of the qbX 174/pC 194 superfamily are characterized by three conserved motifs, 1, 2, and 3, which are present in the same arrangement from the N- to C-terminal.~5 The motif 3 contains either one or two active tyrosines involved in the cleavage and reactions of transphosphorylation. The two putative proteins encoded by pGT5 (orfl and orf2, Fig. 1A) have no detectable similarity 15 T. V. Ilyina and E. V. Koonin, Nucleic Acids Res. 20, 3279 (1992). 16 j. S. Yu, and K. M. Noll, J. Bacteriol. 179,7161 (1997). J7 N. R. Hackett, M. P. Krebs, S. DasSarma, W. Goebel, U. L. RajBhandary, and H. G. Khorana, Nucleic Acids Res. 18, 3408 (1990).
[ 16]
Rep75
FROM
Pyrococcus abyssi
195
with other proteins in databases using search programs such as FASTA or BLAST. However, the three motifs characteristic of the q~X174/pC194 Rep superfamily can be detected by visual inspection in the putative protein of 75 kDa encoded by orfl.1 Moreover, an 11 nucleotide sequence, identical to the pC 194 dso, is present in the pGT5 sequenceJ These two observations suggested that pGT5 replicated via the RC mechanism and, as a result, could be another archaeal member of the qbX 174/pC 194 superfamily.15 This prediction has been confirmed by biochemical analyses. It was shown that: (1) cells ofP. abyssi contain a single-stranded form of pGT5 that has the characteristic of a RC replication intermediate I; (2) the purified Rep protein of pGT5 (Rep75) exhibits a highly thermophilic and specific nickingclosing (NC) activity on single-stranded oligonucleotides containing the pGT5 dso sequence 18; and (3) replacement of the only tyrosine located in motif 3 by a phenylalanine abolishes this activity.19 In addition to its expected activities (NC), Rep75 exhibits an unusual site-specific nucleotidyl-terminal transferase (NTT) activity, never described before for a Rep protein. 18This transfer occurs only at the 3' end of the nicking site with an adenine (or a deoxyadenine) nucleotide monophosphate, and it was proposed that this NTT activity plays a role in the regulation of pGT5 replication. 19 The protein Rep75 can be overproduced in Escherichia coli and purified to near homogeneity. The method used, as well as the activity tests, is described in detail here and could be relevant to the study of other hyperthermophilic Rep proteins. It is still not known if Rep75 corresponds to the physiological form of the pGT5 Rep protein., since the orfl of pGT5 contains three in-frame initiation codons (Fig. 1), and thus could encode for three putatives Rep proteins of 75, 66, and 50 kDa, respectively (Rep75, Rep60, and Rep50). In particular, since all Rep proteins from bacterial plasmids and viruses, as well as Rep proteins from geminiviruses, are smaller than Rep75 (in the range of 20 to 40 kDa), the physiological form of pGT5 Rep might have been Rep50 or Rep60. This chapter also describes the purification of the Rep50 protein expressed in E. coli and its biochemical characterization. Results have shown that Rep50 cannot be the physiological form of pGT5 Rep and provide novel information about the mechanism of Rep75 activity. Unfortunately, the rep60 gene has yet to be cloned in E. coli, because of the instability of this DNA fi'agment in pET3b vector. P u r i f i c a t i o n of R e c o m b i n a n t p G T 5 R e p 7 5 P r o t e i n
Cloning of rep75 Gene The Rep proteins of RC plasmids are usually present in very low copy number in the host cell. In bacteria, these proteins have been successfully purified from 18S. Marsin and E Forterre,Mol. Microbiol.27, 1183(1998). 19S. Marsin and P. Forterre,Mol. MicrobioL33, 537 (1999).
196
NUCLEICACIDMODIFYINGENZYMES
[1 6]
host cells only when it has been possible to take advantage of mutations that upregulate Rep expression in vivo. In the absence of such system, Rep proteins should be overproduced using expression vectors. The bacteriophage T7 expression system (pET3b plasmid from Stratagene, La Jolla, CA) is very efficient to obtain high amount of the pGT5 Rep75 proteins in E. coli. In this plasmid, the gene of interest is under the control of the bacteriophage T7qbl0 promoter and located downstream of a strong ribosome binding site. T7 RNA polymerase is provided by isopropylthiogalactoside (IPTG) induction of the T7 RNA polymerase gene under control of the lac repressor in a defective integrated lambda bacteriophage. The rep75 gene has been cloned into the pET3b plasmid from PCR products amplified from the pGT5 plasmid using Vent polymerase ~8 (New England Biolabs, MA). The recombinant plasmid is called pETRep75.
Cell Culture and Induction
Cells of E. coli BL21(hDE3) pLysS, carrying the plasmid pETRep75, are grown at 37 ° in L-broth containing 100 Ixg/ml ampicillin and 25 Ixg/ml chloramphenicol with vigorous aeration up to an absorbance of 0.6 at 600 n m (A600). The Rep75 protein is induced by addition of 0.5 mM IPTG followed by growth for 3 or 4 hr up to an A600 close to 1. After induction, Rep75 can represent up to 30% of E. coli proteins (compare TO and T4 on Fig. 2). The cells are then harvested by centrifugation without previous chilling, to prevent lysis, and kept frozen at - 2 0 ° .
(kDa) M 205 116 66 55 45 36
TO
T4
S
Wl
W2
IB
PC
29 24 19 FIG.2. SDS-PAGEanalysisof Rep75 purification. The proteins were analyzedby electrophoresis usinga 10%gel stainedwithCoomassieBrilliantBlueandphotographed.M, molecularsizemarker;TO, E. coli crude extract of uninducedculture; T4, E. coli crude extract after4 hr of induction; S, soluble proteins after sonication;W1 and W2, first and second wash, respectively,in buffer C of insoluble proteins; IB, aggregatedRep75resolubilizedin bufferD; PC, Rep75elutionfromthe phosphocellulose column.
[ 16]
Rep75 FROM Pyrococcus abyssi
197
Purification of lnclusion Bodies Cells from 100 ml culture are resuspended at an A60o of 30 in 3 ml of ice-cold buffer A300 (25 mM Tris-HC1, pH 8.8, 300 mM NaC1, 2 mM dithiothreitol, and 1 mM EDTA) and broken by sonication. Three ml of ice-cold TE buffer is then added, and the viscosity due to DNA is reduced by incubation with 10 txg/ml DNase I in the presence of 8 mM MgC12 for 1 hr at 4 °. Three ml of ice-cold buffer B [ 10 mM Tris-HC1, pH 8.8, 100 mM NaC1, 1% Nonidet P-40 (NP-40), 0.5% deoxycholate, and 1% Triton X-100) is added and mixed to solubilize membranes. Inclusion bodies containing Rep75 are collected by centrifugation at 10,000g for 10 min. The supernatant contains very little detectable soluble Rep75 protein (compare fractions T4 and S in Fig. 2). The pellet is washed twice in ice-cold buffer C (50 mM Tris-HC1, pH 8.8, 500 mM NaC1, and 1 mM EDTA). Part of Rep75 is lost in the first wash (W1), but this step eliminates some contaminants (compare fractions W1 and IB, Fig. 2). The insoluble material present in the pellet is solubilized in 1 ml of buffer D (10 mM Tris-HC1, pH 8.8, and 6 M guanidinium hydrochloride) (fraction IB, Fig. 2). Rep75 is completely solubilized by this procedure, whereas it is only partly solubilized in 8 M urea.
Renaturation and Purification of Solubilized Rep 75 Fraction IB is diluted in buffer D down to a protein concentration of 0.1 mg/ml. Solubilized proteins present are renaturated by continuous dialysis overnight at 4 ° against buffer A200 (buffer A300 with 200 mM NaC1 instead of 300 mM). A large amount of protein precipitates during this step (see comments) and the aggregate is eliminated by centrifugation for 10 min at 15,000g. The supernatant is loaded on a 2 ml P11 cellulose phosphate (Whatman, Clifton, NJ) column equilibrated in buffer A200 and then washed with five volumes of buffer A300. Rep75 is eluted with 5 ml of buffer A450 (buffer A300 with 450 mM NaC1 instead of 300 mM) (fraction PC, Fig. 2). Rep75 can be stored in this buffer with 0.08% sodium azide for 1 month at 4 °, and for at least 1 year at - 8 0 ° without loss of activity.
Comments about Rep75 Purification Soluble recombinant Rep75 protein could not be obtained by changing the conditions of induction, such as growth temperature or IPTG concentration. A purification procedure was therefore designed that takes advantage of Rep75 insolubility, using the isolation of inclusion bodies as an enrichment step. There were significant amounts of Rep75 in inclusion bodies, corresponding to about 5 mg per 100 ml of culture. Rep75 was recovered after solubilization of the inclusion bodies followed by renaturation of the protein. During the renaturation step, 70 to 80% of the protein aggregated. This percentage increases if the initial Rep75
198
NUCLEIC ACID MODIFYINGENZYMES
[16]
concentration is more than 0.1 mg/ml. Aggregation also occurs in the phosphocellulose column if the column volume is less than 1 ml for 1 mg of soluble protein. Moreover, after elution, less than 0.25 mg/ml of Rep75 protein was obtained, and it was not possible to concentrate Rep75 further. Nevertheless, between 0.5 mg and 1 mg of pure recombinant Rep75 could be obtained routinely per 100 ml of culture. Rep75 is >95% homogeneous as determined by Coomassie Blue stained SDS-PAGE. Rep75 preparations obtained after denaturation/renaturation could a priori contain both inactive (incorrectly folded) and active proteins. This is likely, in view of the amount of protein required to get in vitro activities (see below). It is not possible to perform quantitative enzymatic analysis with recombinant Rep75 since it was not possible to determine the proportion of active protein in the final fraction (PC, Fig. 2). Preparations sometimes contained low amount of polypeptides that have molecular weights, slightly lower than those of Rep75 and that cross-react with Rep75 antibodies (data not shown). These proteolytic products of Rep75 are as thermostable as full-size Rep75 protein (Fig. 3A). Figure 3A shows that only 60% of recombinant Rep75 remained soluble when the purified protein is incubated for 1 hr at 100 °. However, the protein preparation retains activity and its level remains constant (this is the case, for example, for the nicking activity illustrated in Fig. 3B). This suggests that the polypeptides that disappear during incubation at 100 ° are those that have not been correctly refolded during the renaturation step of the purification procedure. One can thus conclude that correctly folded and active Rep75 proteins are fully stable for at least 1 hour at 100 °.
A Incubation time at 100° 0 5 15 30 60 !
T
B Preincubationtime ofRep75at100°(rain) 0 5 10 15 30 60
i i
*I6L-9R
Rep75~
*16L
FIG.3. Rep75 thermostability. (A) Rep75 remains soluble after incubationat 100°. Purified Rep75 was incubated at 100° during the indicated time and centrifuged for 10 min at 15,000g to eliminate insoluble material. Soluble Rep75 proteins were then loaded on a 10% SDS-PAGE.The gel shown was stained with Coomassie Brillant Blue. (B) Rep75 remains active after incubation at 100°. The Rep75 protein incubated at 100° during the indicated time was used in the nicking test. The labeled oligonucleotide substrate *16R-9Land its cleavage product *16L are indicated.
Rep75 FROM Pyrococcus abyssi
[ 16]
199
Recombinant pGT5 Rep50 Purification The rep50 gene has been cloned in the pET3 plasmid, using the same strategy as for the rep75 gene. However, in contrast to the plasmid pETRep75, the plasmid harboring the rep50 gene (pETRep50) is unstable in the BL2 IpLysS strain and is lost during the culture. To express Rep50, several colonies of transformed cells are thus selected on plates and used immediately as inoculum for the overproducing culture. Rep50 was then expressed, as described for Rep75. The protein Rep50 is also insoluble and can be recovered from purified inclusion bodies. Rep50 is solubilized in I ml of buffer D and renatured simply by a 10-fold dilution in buffer A300. Some aggregated material is then removed by centrifugation for I0 min at 15,000g. In contrast to Rep75, Rep50 does not bind to a phosphocellulose column; however, the protein Rep50 is at least 90% pure (similar to the degree of Rep75 purity in fraction IB). Approximately 0.5 mg of Rep50 protein is obtained from 100 ml of culture.
Assay Method The activity of Rep proteins from RC plamids can be assayed on the basis of their ability to support in vitro replication initiation of RC plasmids. This is very tedious since it requires one to set up a complete in vitro system with host proteins. The basic function of Rep proteins is nicking-closing activity, which is usually specific for DNA-containing the dso plasmid sequence. This activity can be detected on supercoiled plasmid as a topoisomerase activity, or on single-stranded oligonucleotides containing the dso nicking site. The latter facilitates better analysis of the reaction, since it is possible to work with small oligonucleotides (less than 50 nucleotides) of defined sequence and to test either nicking or closing activity, as well as the novel terminal transferase activity discovered in Rep75. In tests for the nicking-closing activity of Rep75 and Rep50, it was noted that oligonucleotides as short as 15-25 nucleotides could be used. The oligonucleotides substrates
TABLE I OLIGONUCLEOTIDES USED IN ACTIVITY TESTSa Name
Sequence
Size
16L-9R 9L- 16R 16L
51-CGTTGGGTTTATCTTG/ATATATCCA-3 ~
25 nt 25 nt 16 nt
5'-TTTATCTTG/ATATATCCACAACCAA-3 ~ 51-CGTTGGGTTTATCTTG
a The characters in boldface type indicate the conserved nucleotides between the putative dso pGT5 sequence and the dso sequence from pC194. The slash indicates the cleavage site.
200
NUCLEIC ACID MODIFYING ENZYMES
[ 16]
routinely used are presented in Table I. They are radiolabelled at their 5' end using [~_32 P]ATR To study the affinity of Rep75 or Rep50 proteins to DNA, the gel shift technique was used, which turned out to be suitable for following binding of the protein on both single-stranded or double-stranded oligonucleotides.
Nicking-Closing For nicking-closing reactions, 20 fmol of the indicated oligonucleotide is incubated at 105 ° with Rep75 in 10 txl of buffer R (50 mM HEPES-HC1, pH 8,200 mM Sodium glutamate, 1 mM DTT, 5 mM MnCI2, 0.1% Triton X-100, 1 mM EDTA, and 50 txg/ml BSA). For ligation tests, the incubation is performed in two steps, first 5 min at 105 ° and then 5 min at 75 °, because of the different temperature requirement between nicking and closing activities (see below). Reactions are stopped on ice by addition of EDTA 25 mM and incubation with 15 txg of proteinase K for 30 min at 55 °. Five ~1 of loading buffer is then added (50 mM Tris-borate, pH 8.3, 80% deionized formamide, 1 mMEDTA, 0.1% xylene cyanol, and 0.1% bromphenol blue). The reaction products are separated on a 20% polyacrylamide (20 : 1) gel containing 8 M urea. Autoradiograms are performed and quantified by scanning the gel with a Phosphoimager ImageQuant.
Nucleotide Terminal Transferase 16L oligonucleotide (Table I), harboring only the left part of the nicking site, is radiolabeled and used in NTT tests. The assay is performed at 75 ° in the same buffer as described for the NC test but with 1 mM of ATP or dATE
Gel Shift Twenty fmol of 5'-radiolabeled single-stranded or double-stranded oligonucleotides is incubated with 1.3 pmol of the Rep protein (protein to DNA molar ratio of 60 : 1) for 5 min. The reaction is stopped by addition of 25 mM EDTA and 5 txl of loading buffer (30% glycerol, 0.25% xylene cyanol, and 0.25% bromphenol blue). The products are then loaded on a 8% polyacrylamide gel (30: 1) in 5% glycerol, 0.25× TBE, and run in 0.25× TBE at room temperature. Incubation temperature is not important for migration under native conditions, and no differences are observed when protein and DNA are previously incubated together either at 105 ° and 4 ° . C o m m e n t s a b o u t R e p 7 5 Activities
Rep75 Nicking-Closing Activity Figure 4 illustrates Rep75 nicking-closing activities on single-stranded oligonucleotides harboring the cleavage sequence of pGT5 dso. The 5' radiolabeled *16L-9R is cleaved by Rep75, so that only the *16L nicking product can be
Rep75 FROM Pyrococcus abyssi
[16]
Reaction type
Nicking I
201
Nleking/Closln 8 NTT I I
II
I
*16I_,-16R *9L-16R
*16L-A *I6L
*9L FIG.4. In vitroRep75 activities. Results of nicking, nicking--closing,and NTT tests were run on a polyacrylamide gel and autoradiographed. The radiolabeled oligonucleotides,used as substrates, are indicated on the upper part. The oligonucleotidesdetectable on the gel are indicated on the sides of the figure. M, markers. detected. The same activity is obtained with the *9L-16R oligonucleotide, which is cleaved to give the *9L product. When the two substrates *16L-9R and *9L16R are incubated together with Rep75, the two nicking products are obtained and the closing product *16L-16R can be detected (the ligation efficiency is too weak here to observe the *9L-9R closing product). A *16L-16R religation product is also obtained when *9L-16R is incubated with 16L and Rep75. No religation is observed when the test is performed with oligonucleotides containing only the right or the left part (16L and 16R oligonucleotides) of the nicking site (data not shown). This is because Rep75 should activate the DNA by nicking and covalent fixation before the religation step at the 3' end. TM
Rep 75 NTT Activity In the presence of ATP or dATE Rep75 exhibits an unusual site-specific nucleotidyl transferase activity, i.e., it transfers one AMP or dAMP to the 3'OH extremity of an oligonucleotide containing the left part of the nicking site. Figure4 illustrates an experiment in which the *16L oligonucleotide (Table II) is transformed into a 17-mer long oligonucleotide (* 16L-A) by this activity. The
202
NUCLEIC ACID MODIFYINGENZYMES
[1 6]
T A B L E II ACTIVITY COMPARISON BETWEEN REP75 AND REP50 a D N A binding
Source Protein Rep75 Rep50
Activities Nicking + +
ssDNA
Closing + .
NTT
Left part
Rightpart
Left pan
Rightpan
+
+
nd
+ nd
+ .
.
dsDNA
.
The activityassayswere performedunder the same conditionswith the two proteins. DNAbinding was performed by gel shift assay as described in the text, on oligonucleotidesingle-stranded (ss) DNA or double-stranded (ds) DNA, these substrates containing the left part or the right part of the nicking site. nd, Not determined.
transfer is slightly better with dATP than with ATP as substrate (Fig. 5C), and the activity is already maximal at 50 R~M. The NTT activity is nucleotide specific, since Rep75 cannot use (d)CTP, (d)GTP, (d)TTP, AMP, ADP, or AMP-PNP. The NTT protein active site at least partly overlaps the NC active site, as an arginine located in the motif 3 of Rep75 is essential for both ligation and NTT activities. However, the nicking and NTT activities can be uncoupled in vitro, since the active tyrosine in motif 3 is dispensable for the NTT activity, and the arginine of motif 3 is dispensable for the nicking activityJ 9 The NC and NTT activities of Rep75 reach equilibrium after 15 min of incubation. Optimal results are obtained when Rep75 is added at a very high protein to DNA molar ratio (60 : 1), Is indicating the low activity of the protein preparation. However, this is not unusual for this type of protein. In nicking assays, one never observes more than 50% of cleavage for the conditions studied, whereas it is possible to observe nearly complete transfer in NTT assays. Moreover, it is difficult to obtain the same specific activity for different protein preparations, probably due to the denaturation step in the purification process.
Comparison of Different Activities of Rep75 The different Rep75 activities are optimal at different assay conditions. The most important and surprising difference is their temperature optima. Figure 5A shows that NTT activity is optimal at 75 °, whereas the nicking activity increases with temperature at least up to 105 ° (verified in a test tube using a thermoprobe). The closing activity is also known to be optimal around 85 ° . 19 The reason for these differences is presently unclear. Another important observation is the difference in the divalent cation requirement between nicking and NTT activities (Fig. 5B). The two activities are optimal
[16]
Rep75
FROM
Pyrococcus abyssi
203
A actlvit', %
activity % 100
60'
transfer % "
75-
80 • 60-
40.
40. 25"
20 20"
0~ 35 45 55 65 75 85 95 105 Incubation temperature
0
2,5
5 7,5 mM cation
10
,
,
//
0 50 100
1000 nucleotide
FIG. 5. Different characteristics of Rep75 activities. (A) Temperature dependence of Rep75 activities. Percentage activity corresponds to the percentage of modificated oligonucleotides, appearance of cleavage product in the case of nicking test (black squares), and apparition of transfer product in the case of NTT assay (white squares). (B) Effect of variation in Mg 2+ and Mn 2+ concentration on Nicking and NTT activities. The activities assays were performed in presence of Mn 2+ (circles) or Mg 2+ (squares). NTT activity (in black) and nicking activity (in white) were quantified as presented in A. (C) Effect of variation in ATP and dATP concentration on NTT activity. The NTT assays were performed for different concentrations of ATP (open circles) or dATP (black circles).
for 5 mM of MnC12. However, the NTT activity is more specific for this salt, since only 25% of the nicking activity is conserved when MnC12 is replaced by MgC12. On the other hand, the NTT activity cannot be observed at all in the presence of MgC12.
Comparison between Rep75 and Rep50 Partially purified Rep50 and Rep75 were compared (Table II). This makes sense since partially purified Rep75 (fraction IB, Fig. 2) exhibits the same activities as the completely pure protein (fraction S). Rep50 can perform site-specific DNA cleavage and its nicking activity is optimal at 105 °, as in the case of Rep75. However, unlike Rep75, Rep50 does not have closing or NTT activities. This clearly indicates that Rep50 cannot be the native protein produced by pGT5 in vivo. The disappearance of the ligation and NTT activities in Rep50 can be explained by the inability of this truncated protein to recognize single-stranded DNA. Indeed, unlike Rep75] 8 Rep50 cannot bind single-stranded DNA fragment harboring the left part of the nicking site, as indicated by gel retardation experiment (Table II). Rep50 could have lost these properties because the single-stranded DNA binding site of Rep75 is located in its N-terminal region, which has been removed from Rep50, or because the absence of this region induces an incorrect protein folding. This suggests that the nicking activity (conserved in Rep50) does not involve the
204
nicking site
5'
[16]
NUCLEIC ACID MODIFYING ENZYMES
Ot ~
+Rep75
O
ct 5'
Y
non covalent fixation
m,,._ 5'
local denaturation
3' 5'
| ~ nicking, covalent fixation
O
3'
~: ~i~i¸ .~a closing
5'
non covalent
interaction FIG. 6. Model of Rep75 origin recognition, a is the left part of the nicking site, 13 is the right part of the nicking site. The model is explained in the text.
strong interaction detected by gel shift between single-stranded DNA and Rep75. In contrast, this interaction should be essential for the closing and NTT activities. Comparison between Rep75 and Rep50 suggests a mechanism for origin recognition by Rep75 (Fig. 6). Rep75 first interacts with the right part of the nicking site (double-stranded) and induces the local melting of DNA. This is similar to the model for recognition of ~X174 dso by gpA, which has been proposed by Baas and Jans. 2° The single-stranded DNA region thus produced is then cleaved by Rep75, which remains covalently linked to the 5' end. The covalently linked protein could then interact with the left part (still single-stranded) of the nicking site and religates DNA. This last step cannot be performed by Rep50, since it is unable to interact with the left part of the nicking site. The proteins Rep75 and Rep50 appear to be an interesting model system to study the mechanism of action of RC Rep proteins, in general. Overproduction of these proteins could pave the way for structural studies on an exciting new family of enzymes that can perform different types of DNA manipulations (cleavage, ligation, and terminal transfer) using a single active site. Acknowledgments This work was supported by grants from the Association pour la Recherche contre le Cancer and the European Community Programm Cell Factory (BIO4-CT 96-0488).
20 E D. Baas and H. S. Jansz, Curr. Top. Microbiol. lmmunol. 136, 31 (1988).
3'