The mechanism of replication of øX174 DNA

J. Mol. Biol. (1975) 99, 107-123

The Mechanism of Replication of ~X174 D N A XII. Non-random Location o f Gaps in Nascent ¢X174 R F H D N A SHLOMO EISENBERG~ BARBARA HARBERS, CHRISTIAN HOURS AND

DAVID T. DEI~HARDT

DeTartment of Biochemistry McGill University Mclntyre Medical Sciences Building 3655 Drummond Stree~ Montreal, Quebec, Canada tt30 1Y6

(t~eceived 20 March 1975, and in revised form 27 July 1975) Nascent $X174 replicative form (RF) D N A purified from phage SX-infeeted Escherichia coli was labelled in a "gap-A11ing" reaction with [~-s2P]d_NTPs using phage T4 D N A polymerase a n d D N A ligase. Three different methods of analysis indicated t h a t the gaps were n o t r a n d o m l y located in either the viral or t h e c o m p l e m e n t a r y strand. (1) R F t D N A labelled with asp in t h e gaps was t r e a t e d simultaneously with t h e restriction endonucleases from Hemophilus influenzas and Hemophi~us aegyptlu8 a n d t h e resulting fragments separated b y electrophoresis on a g a r o s e p o l y a c r y l a m i d e gels. The distribution of the 32p in the fragments of t h e gaplabelled R F was c o m p a r e d to t h e distribution of 32p present in t h e fragments of uniformly-labelled ~X R F D N A ' a n d found to be different. (2) The viral a n d c o m p l e m e n t a r y strands of the gap-labelled R F were separated b y reannealing in t h e presence of excess viral strands, followed b y h y d r o x y a p a t i t e c h r o m a t o g r a p h y ; the separated strands were t h e n digested with formic acid/ diphenylam~n e. The distribution of pyrimidine oligonucleotides in each s t r a n d was determined b y ionophoresis-homochromatography a n d found to differ from the distribution observed when the entire s t r a n d was uniformly labelled. (3) The n u m b e r of gaps in the population of R F molecules under s t u d y was determined from t h e a m o u n t of sup acquired from [7-s2p]ATP in a reaction catalyzed b y polynucleotide kinase. I n separate reactions t h e a m o u n t of each of t h e four deoxynucleoside monophosphates incorporated in the gap-~11ing reaction was determined; combination of this information with t h e n u m b e r of gaps indicated t h a t the average gap in b o t h the viral a n d the c o m p l e m e n t a r y strands was 13 to 16 nucleotides long. The proportions of t h e four nueleotides incorp o r a t e d into the gaps in the viral a n d c o m p l e m e n t a r y strands were different from t h e proportions found in the complete viral a n d c o m p l e m e n t a r y strands, a n d were biased in favour of t h e pyrimidine nueleotides in b o t h eases. These observations suggest t h a t synthesis of all viral D N A during ~X R F replication is initiated a t n o n - r a n d o m l y distributed sites on t h e SX genome. The origin of replication appears to be in t h e Z6b Hemophi~ua aegypti~s restriction endonuclease cleavage fragment a n d to contain t h e unique CeT pyrimidine tract. J" Abbreviations used: RF, Replioative form; R F I is eovalently closed; R F I I possess at least one diseontinuity.

107

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S. E I S E N B E R G E T AL. 1. I n t r o d u c t i o n

The life cycle of bacteriophage ~X174 can be divided into three different phases: (1) Parental replicative form (RF) formation, which is characterized b y the synthesis of the strand complementary to the infecting circular viral DNA. The enzymology of this process has been under intensive investigation recently in an in vitro soluble system (Schekman etaL, 1974; Wickner & Hurwitz, 1974). (2) Semiconservative replication of the R F t, as a result of which 20 to 50 R F molecules accumulate in the cell (Sinsheimer, 1968). (3) Asymmetric displacement of the viral strands from R F molecules, circularization of the single-stranded viral DNA b y an unknown process (Miller & Sinsheimer, 1974), and immediate encapsulation of the DNA int~) phage particles. Previously we reported a structural analysis of R F molecules found in the cell during R F replication. The R F I I I)NA has a circular structure with at least one discontinuity in one of the strands. The discontinuity in newly-synthesized R F is a gap, and its closure requires the combined action of a DNA polymerase and DNA ligase (Schekman etaL, 1971). The gaps are asymmetrically distributed between the two strands of the R F I I DNA. There appears to be one gap in the viral strand located in a specific region, the region of cistron A on the CX genetic map, whereas the gaps in the complementary strand are located a~ m a n y sites on the ~ X genome (Eisenberg & Denhardt, 1974a,5). I t was concluded t h a t the complementary strands are synthesized discontinuously; in contrast, the viral strands are synthesized continuously star~ing in the region of gone A. Initiation of a " r o u n d " of R F replication is also initiated in the cistron A region on the ~ X genetic map (Baas & Jansz, 1972; Godson, 1974). Here we present experiments which show t h a t the gaps in the complementary strand of nascent R F I I DNA are not randomly distributed over the ~X genome, and we con6rm the previously reported non-random location of the gap in the viral strand.

2. Materials and Methods (a) Bacteria and bacteriophage Escherichia co~i HF4720 polA1 has been described by Schekman et al. (1971). ~X174am3 is a lysis-defective cistron E mutant.

Co) Reagents Deoxynucleoside-5'-triphosphates were purchased from P-L Biochemicals Inc. [methyl8H]thymidine (40 to 60 Ci/mmol), [~.32P]dATP and [~-82PJdGTP with specific activity of 2.4 × 1011cts/mln per pmol were bought from New England Nuclear Corp. [r-a2P]ATP with a specific activity of 2× 1011 cts/mln per pmol was prepared by the method of Glynn & ChappeU (1964). [~-32P]dCTP and [~.auP]dTTP with specific activity of 5 × 101° to 2.2 × 1011 cts/min per pmol were prepared by the method of Symons (1969). Hydroxyapatite (HTP grade) was a Bio.Rad product. (c) Enzymes Pronase, grade B, was bought from Calbiochem. Pancreatic RNase and bacterial alkaline phosphatase were purchased from Worthington. Prior to use, 2 nag of pancreatic RNase were dissolved in I ml of 30 m~-sodium acetate buffer (pH 5-5) and heated at 85°C for 20 mln. Commercial E. coli bacterial alkaline phosphatase was further purified on DEAE-eellulose columns as described by Weiss et aL (1968). T4 polynucleotide kinase was purified by a slightly modified form of the procedure described by Richardson (1965). The enzyme was eluted from the phosphocelluloso column using a linear gradient of 0 t Please see footnote on p. 107.

NON-RANDOM GAP LOCATION IN NASCENT ~X RF

109

to 0"5 ~-KC1 in 0"05 m-potassium p h o s p h a t e buffer (pH 7.5). T4 D N A polymerase was purified through t h e h y d r o x y a p a t i t e stage (Goulian eta/., 1968), a n d T4 D N A ligase was purified as described b y Weiss (1971). None of t h e enzymes described above h a d a n y detectable endonuclease a c t i v i t y as j u d g e d from a sedimentation velocity analysis in sucrose gradients of enzyme-treated CX R F I a n d single-stranded circular D N A . T h e restriction endonuclease from Hemophilus influenzae R d and Hemophilu8 ae.gypti~ were purified as described b y S m i t h & Wilcox (1970) a n d Middleton et al. (1972), respectively, w i t h t h e modifications described b y Eisenberg & D e n h a r d t (1974a). (d) Preparation of R F I I DlqA F o u r 500-ml cultures of E. coli 4720 were grown in mT3X]:) m e d i u m (Dcnhardt, I969) with aeration to a b o u t 2 × 108 cells/ml a n d infected with ~bX174am3 a t a multiplicity of 5. Six rain after infection [3H]thymidine was a d d e d (I pCi/ml) a n d 3 rain later incorporation was t e r m i n a t e d b y pouring t h e culture into an equal volume of a solution containing either: (1) 75~/o ethanol, 20 m ~ - s o d i u m acetate (pH 5"5), 2 r n ~ - E D T A a n d 2 % phenol a t room t e m p e r a t u r e or (2) 0-1 ~-NaC1, 10 m~-Tris-HC1 (pH 8-1), 1 mM-EDTA a n d 20 n ~ K C N a t 0°C. The cells were collected i m m e d i a t e l y b y centrifugation in a Sorvall RC3 centrifuge for 30 raln~ 4°0 a t 4400 revs/min. The cells were resuspended in 160 m l of 0.05 ~ T r i s - H C l (pH 8.1) a n d lysed b y incubation w i t h 0-7 lysozyme/ml a n d 20 r a ~ - E D T A a t 37°C for 3 rain. Sodium dodecyl sulphate a n d Pronase (20 m g / m l in 0.25 ~-Tris-HC1 (pH 8.1), p r e i n c u b a t e d a t 37°C for 3 h) were subsequently a d d e d to a final concentration o f 1% a n d 0.8 mg/ml, respectively, and t h e incubation continued for another 8 to 12 h. A 5 ~-NaC1 solution was then a d d e d a n d m i x e d in gently b y r o t a t i n g the t u b e to give a final concentration of 1 ~-NaCI. A co-precipitate o f E. coli D N A with sodium dodecyl s u l p h a t e was allowed to form a t 0°C for 8 to 12 h a n d was r e m o v e d b y centrifugation a t 34,000 revs/min, 0°C for 3 h in t h e t y p e 35 rotor in a Beclrra~.n L2-65B ultracentrifuge. The s u p e r n a t a n t was e x t r a c t e d with phenol (water-saturated) a n d t h e viral nucleic acids p r e c i p i t a t e d with sodium acetate-isopropanol as described b y Schekman e t a / . (1971). T h e nucleic acids were collected b y eentrifugation in t h e Sorvall RC2 centrifuge, GSA rotor, 10,000 revs/min, 30 rain a t 0°C. The pellet was dissolved in 7 m l of 50 raM-Tris.HC1 (pH 8.1)-40 mM-EDTA buffer. Pancreatic R N A a s e was a d d e d to a final concentration o f 250 pg/ml a n d t h e solution i n c u b a t e d for 60 rain a t 37°C. ~X R F was further purified b y centrifugation t h r o u g h a n e u t r a l sucrose gradient (SW 27 rotor, 15 h, 24,000 revs/raln 15°C, B e c k m a n L2-65B ultracentrifuge). 1-ml fractions were collected from t h e t o p o f t h e gradient with a Buchler Auto-Densi Flow. The 3H-labelled 16 S R F IX D N A fractions were pooled, p r e c i p i t a t e d with sodium acetate-isopropanol, a n d centrifuged to equilibrium in a n e u t r a l CsC1 density gradient (1.71 g CsC1/ml) in order to remove residual conf.~m~ating R N A . (e) Determination of th6 average size and base.compoai~on

of gaps in R F I I DlgA The average size of t h e gaps was determined in t h e R F I I D N A t h a t could be converted in vitro to a n R F I structure. I n order to m a k e this m e a s u r e m e n t i t was first necessary to determine t h e n u m b e r of gaps in the D N A ; this was accomplished b y labelling the 5' end (the R F I I h a d previously been t r e a t e d with sufficient alkaline phosphatase to remove all t e r m i n a l phosphates) o f t h e gaps with [r-s2P]ATP using T4 polynucleotide kinase. T h e n u m b e r of nueleotides t h a t were required to completely fill in t h e gaps was deduced from t h e a m o u n t of [3~'P]dNMP incorporated in a reaction with T4 DlqA polymerase, T4 D N A ligase a n d all four [~-a2P]dNTPs. The incorporation of s2p into t h e R F I was normalized to the a m o u n t of aH uniformly present in the R F . Thus, t h e average size of the gap =

moles of [s2P]dNMP incorporated moles of 5' ends present

(i) Bacterial alkaline p h o s p ~ e reaction The R F I I D N A purified from 2 1 of ~X-infected E . coli 4720 cells was t r e a t e d with bacterial alkaline phosphatase a t 65°C for 30 rain. A t 10-raln intervals 1-5 units of alkallne phosphatase were a d d e d t o a reaction m i x t u r e containing 10 m~-Tris.HC1 (pH 8"I) a n d

110

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~ X R F I I D N A (10 to 20/Lg) in a t o t a l volume of 200/~1. A t t h e end of t h e incubation period t h e R F IT D N A was repurified b y centrifugation on n e u t r a l sucrose gradients (SWS0.1, 15°C, 50,000 tees/rain, 3 h). (ii) T g polynucl~otide kinase reaction The reaction miYture, in a t o t a l volume of 50 ~1, contained 0.1 ~-Tris.HC1 (pH 7.6), 10 m~-MgC12, 10 m~t-dithiothreitol; 2 n m o l of [~-3aP]ATP or unlabelled A T P , R F I I D N A (1 to 1-5 /~g) a n d T4 polynucleotide kinase (an a m o u n t sufficient t o achieve 20 to 25% conversion of R F I I to R F I in t h e D N A polymerase plus D N A ligase reaction). The reaction was incubated for 90 rnJn a t 37°C.

(fii) Conversion of R F I I to R F I The T4 polynucleotide kinase reactions (above) were supplemented with 2 nmol each of d T T P , dCTP, d G T P , dATP, 20 nmol of A T P , 0.02 units of T4 D N A polymerase a n d 0-6 units o f T 4 D N A ligase in a final volume of 75/~1. The reaction was allowed to continue for a n additional 80 rain before i t was t e r m i n a t e d b y t h e a d d i t i o n o f 1 2 0 / d of a solution containing 0.1 ~ - E D T A , 0"3 ~ - N a O H a n d 4 /~g calf t h y m u s D N A carrier. The entire reaction mixture was l a y e r e d on an alkaline sucrose gradient a n d the in v/tro-formed R F I isolated after centrifugation for 60 rain in t h e S W 50.1 rotor a t 50,000 revs/mln, 15°C, in the B e c k m a n L2-65B ultracentrifuge. Those reaction mixtures containing R F I I D N A with 82p label a t the 5' ends of t h e gaps (to determine t h e n u m b e r of gaps present) were supplemented with unlabelled d_NTPs. The four separate reaction mixtures containing R F H D N A with s1p a t t h e 5' end of the gaps (to q u a n t i t a t e t h e incorporation into gaps) were supplemented similarly b u t each with a different [a.s2P]dNTP. (iv) Determination of the amour~ of 3ap label incorporated into complementary and viral strands of the in vitro-fornm~ R F Z R F I D N A obtained as described in section (e) (iii), above was dialysed against a 0.1 ~-Tris.HC1 (pH 7.6), 0.1 M-NaC1 a n d 2 m ~ - E D T A buffer. Complementary a n d viral strands were s e p a r a t e d in a competition-annealing experiment performed as described b y Eisenberg & D e n h a r d t (1974a). The single-stranded D N A (containing the labelled viral strand) a n d double-stranded D N A (containing t h e labelled c o m p l e m e n t a r y strand) were fractionated on h y d r o x y a p a t i t e columns. The fractionation on h y d r o x y a p a t i t e columns was performed essentially b y t h e m e t h o d of Wilson & T h o m a s (1973). A 0-5 m l h y d r o x y a p a t i t e column was p r e p a r e d in a P a s t e u r p i p e t t e a n d washed with 10 m ~ - p o t a s s i u m phosphate buffer (pH 6.8). The D N A samples were applied to the columns which were t h e n washed successively with 10 ml of 10 m ~ - p o t a s s i u m p h o s p h a t e buffer (pH 6"8), 10 ml of 0-14 ~ - p o t a ~ i u m phosphate buffer (pH 6-8), a n d I0 mt of 0-3 M-potassium p h o s p h a t e (pH 6.8). 1-ml fractions were collected directly into scintillation vials, to which 20 ml of t o l u e n e - T r i t o n X l 0 0 scintillation fluid (Sebring etal., 1971) were added. The fractionation was performed a t room temperature. Control experiments with 3H-labelled single-stranded D N A a n d 82P-labelled R F verified t h a t single-stranded a n d double-stranded D N A s were eluted in the 0.14 ~ a n d 0.3 ~-potassitma phosphate washes, respectively. (f) Preparation of 32P-labelled ~X complementary st,rand D N A The s t r a n d c o m p l e m e n t a r y to ¢X174 single-stranded circular D N A was synthesized in vitro in a 250/A reaction miYture containing 5.6/~g ~X single-stranded DNA, 50 m ~ l~T-Tris(hydroxymethyl)-methyl-2-amino ethane sulphonic acid buffer (pH 7.0), 10 m ~ MgCI~, 50 mM-KC1, 1 m~-dithiothreitol, 40 /~g bovine serum albumin, 0-25 m ~ - d A T P , 0.25 m ~ - d G T P , 0"12 m~-[g-32P]dCTP a n d [~-~2P]dTTP with specific activities of 9 × 109 cts/min per/maol a n d 4 × 109 cts/min per/~mol, respectively, 50 ml~-ATP, 3 ~g of deoxyoligonucleotides (prepared as described b y Schekman etal., 1971), 0.12 units of •. cell D N A polymerase I (purified b y t h e procedure of J o v i n eta/. (1969), a gift of G. McFadden) a n d 1.5 units o f T 4 ligase (a gift from A. K a t e ) . The reaction was i n c u b a t e d for 12 to 13 h a t 37°C. The R F was purified b y centrifugation on neutral sucrose gradients (SW 50.1 rotor, 50,000 revs/mln~ 3 h, 15°C in t h e B e c k m a n L2-65B ultracentrifuge). The R F I was

NON-RANDOM GAP L O C A T I O N I N NASCENT SX R F

111

separated from the R F I I in CsC1 equilibrium density gradients containing ethidium bromide as described by Schekman e$ at. (1971). (g) Gap-filling reaction and 8sparation of vira~ and complementary

strands.for pyrimidine tract analysis The gap-~lliug reaction was performed as described by Eisenberg & Denhardt (1974a). The gap regions were labelled with [a-32P]dCTP and [~-z2P]dTTP of 1.1 × I011 cts]min per ~mol and 6.7 × 101° cts/min per ~mol specific activities, respectively. [SH, 32P]RF I I DNA was purified on CsCl-ethidium bromide density gradients to remove R F I. I n order to separate the complementary and viral strands 5 to 10 ~g of [3H, z2P]RF I I DNA (no$ sonicated) were m~Yed with 150 ~g unlabelled viral DNA in a total volume of 200 ~1, containing 0.1 ~-Tris.HC1 (pH 7.6), 0.1 M-NaC1, and 2 m~-EDTA. The reaction m~Yture was incubated at 100°C for 5 rain and placed in a 60°C water bath for 15 h (a Cott sufficient to give complete renaturation). Single- and double-stranded DNA was fractionated on hydroxyapatite as described above. DNA eluted with 0.14 M.potassium phosphate buffer (single-stranded viral strands) and DNA eluted with 0.3 ~-potassium phosphate buffer (complementary strands in duplex DNA) were pooled separately, dialysed extensively against distilled water, and concentrated by precipitation with sodium acetate--isopropanol. (h) Fractionation of pyrimidine oligonucleotides The DNA was depurinated by incubation with 2% diphenylamine in 67% formic acid at 30°C for 18 h according to the procedure of Burton & Peterson (1960). The reaction mixture was extracted three times with ether, dried and redisselved in 5 to 10 #1 of distilled water. The depurination products were fractionated by ionophoresis-homoehromategraphy (Brownlee & Sanger, 1969; Ling, 1972). Samples were applied to a cellnlose-acetate strip (3 cm × 50 cm) together with 2 ~1 of a standard mixture of 3 dyes. Electrophoresis was carried out for 20 to 30 rain at 4.5 kV in an eleetrophoresis buffer containing 4"5% acetic acid, 0.5% formic acid, 5 m~.EDTA and 7 ~-urea. The separated oligonucleotides were then blotted onto DEAE-cellulose thin-layer p l a t ~ (20 e m × 20 cm), and chromatography was carried out at 60°C in a 2% solution of partially hydrolysed yeast RNA (Jay et al., 1974). The fractionated pyrimidine oligonucleotides were first located by autoradiography, the appropriate areas scraped off the DEAE-eellulose plate, and toluone-PPOPOPOP scintillation fluid added to each sample. The amount of radioactivity present was determined in an Intertechnique Multi-Mat scintillation counter. We are aware that a comparison of the distribution of s2p among the pyrimidine oligonucleotides of the uniformly s2p-labelled viral strand and the viral strand containing s2p only in the gaps is not rigorous. This is because in the gap-~ll~ng reaction with T4 DNA polymerase s2p was present only in the pyrimidine nucleoside triphosphates; thus the phosphate at the 3' end of the pyrimidine oligonucleotides derived from the gap-filled molecules cannot contain s2p. Also, the specific activities of the dCTP and dTTP were not identical. However, for the conclusions we wish to draw this absence of strict comparability (which could be corrected for if necessary) is tolerable. I n the case of the complementary strands the comparison is, of course, accurate.

3. Results (a) Distribution among the fragments produced by the combined action of Ttind:~ and Hae restriction enzymes of ssp incorporated into the gaps Nascent [aH]RF I I D N A purified from ¢X-infeeted cells during R F replication was labelled specifically, and to completion, in the gaps with [a-82P]dCTP, using T4 D N A polymerase and T4 D N A ligase as described b y Eisenberg & :Denhaxdt (1974a,b). Evidence t h a t all the gaps were filled in is presented in Figure 1. The presence of gaps t Got is the product of initial concentration and time. :~ Abbreviations used: Hind and Hae, H6moph//us ir~fl~nzae, serotype d, and Hemol~h//~ aegyptiua restriction endonuelease, respectively.

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Fraction no. Fzo. 1. Sedimentation analyses on n e u t r a l sucrose gradients of ~X174 R F I I D N A t r e a t e d w i t h N . crcssa single-strand specific endonuelease before a n d after t h e gap-fil~ng reaction. ~ X R F I I was pulse-labelled i~ ~vo w i t h [SH]thymidine a n d e x t r a c t e d a t 10 rain after infection from E. coli HF4720 a n d described in Materials a n d Methods, section (d). (a) a n d (b) The [SH]RF I I D N A was mixed w i t h uniformly s2p-labelIed ~ X R F I m a r k e r a n d t r e a t e d w i t h N . cra~aa single-strand specific endonuelease a t s concentration of 3 units]ml for 45 m i n a t 37°C using t h e conditions described b y K a t e e~ al. (1973). The s e d i m e n t a t i o n profiles of t h e D N A before a n d after t r e a t m e n t are shown in (a) a n d (b), respectively. Much of t h e [s=P]RF I (75%) was converted to R F I I (as expected, K a t e et al., 1973) a n d t h e [3H]RF H was c o n v e r t e d to R F H I a n d more slow]y-sedimenting species, presumably arising from those molecules t h a t h a d more t h a n one gap. (o) a n d (d) [aH]RF I I molecules prepared as above were reacted w i t h T4 D N A polymerase, T4 D N A ligase, a n d [=-3=P]dCTP as described b y Eisenberg & D e n h a r d t (1974a). The gap-filled [32p, 3H]RF was purified on a n e u t r a l sucrose gradient a n d split into two portions, one of which was t r e a t e d w i t h t h e single-strand specific endonuclease as described above. T h e s e d i m e n t a t i o n profiles of t h e D ~ A before a n d after exposure to t h e enzyme are shown i n (o) a n d (d), respectively. I n b o t h gradients t h e D N A sedimented to t h e position (identified i n parallel gradients) of R F I I (closed circular duplexes m a d e i ~ t~tro u n d e r our conditions also sediment to this position since t h e y do n o t contain superhelieal twists). There was no change in t h e sedimentation properties of t h e R F after exposure to t h e enzyme, suggesting t h a t there were no nuelease-sensitive sites (gaps) in t h e DNA. The high salt (1 ~r-NaC1), n e u t r a l sucrose (5% to 20%) gradients were centrifuged for 3 h a t 50,000 r e v s / m l n in a S W 50.1 rotor in a B e c k m a n L2 65 B ultracentrifuge. Sedimentation is from right to left. 3H, - - $ - - @ - - ; 32p, .. O "" O "'.

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PLATE I. (a) Pyrimidine tracts present in the 3aP-labelled complementary strand. (b) Grid p a t t e r n of 3', 5' p h o s p h o r y l a t e d pyrimidine oligonucleotides. The (tiagram is based on the oligonucleotide p a t t e r n of (a). The position of each oligonucleotide is represented b y a dot (.). Solid lines join oligonucleotides of similar cytosine content, a n d broken lines those of similar t h y m i n e content. The base-composition of each py]'imidine t r a c t was determined according to H a r b e r s e~ al. (1974). (e} Pyrimidine t r a c t s characteristic of t h e gaps found in the c o m p l e m e n t a r y s t r a n d of nascent R F I I DNA. Synthesis of 32P-labelled c o m p l e m e n t a r y strands, gap-labelling, separation of R F I I strands, a n d pyrimidine t r a c t analyses were performed as described in Materials a n d Methods. [facing p. 112

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PLATE II. (a) Pyrimidine tracts present in the uniformly-labelled viral strand. (b) Grid p a t t e r n of 3', 5' phosphorylated pyrimidine oligonucleotides. The diagram is based on the oligonucleotidc p a t t e r n of (a). The position of each oligonucleotide is represented b y a dot (.). Solid lines join oligonueleotides of similar cytosine content, a n d b r o k e n lines join those of similar t h y m i n e content. The base-composition of each pyrimidine t r a c t was determined according ~o H a r b e r s et aZ. (1974). (o) Pyrimidine tracts characteristic of the gap found in the viral s t r a n d of nascent R F I I DNA. Viral strands labelled in t h e gap region were obtained as described in Materials a n d Methods. Uniformly 32P-labelled viral D N A was e x t r a c t e d from 32P-labelled ~X174 phage particles, which were obtained b y the procedure described b y S e h e k m a n et al. (1971).

NON-RANDOM GAP LOCATION IN NASCENT ~X RF

113

in the nascent R F II renders the R F II sensitive to the action of the Neurospora crasaa single-strand specific nuclease ((a) and (b)); after the gaps are filled in the R F II is no longer sensitive to the enzyme ((c) and (d)). l~rther details are given in the legend. The gap-labelled [82p, 3H]RF DNA (I ~ II) was treated with Hind and Hae nucleases simultaneously, and the fragments produced were separated by electrophoresis on agarose-polyacrylamide gels (Fig. 2(a)). Uniformly 89.p. and [SH]thymlnelabelled t X R F DNAs were mixed and treated with the two restriction enzymes in a similar manner (Fig. 2(b)). All the uniformly-labelled fragments in Figure 2(b) had the same 32P/3H ratio. The data shown in Figure 2(a) indicate, however, that the 82P/SH ratio varied significantly in the different fragments from the gap-labelled molecules. This result suggests that the distribution of the gaps in the R F II molecules is not random; however, the alternative interpretation of a non-nnfform distribution of cytosine residues has not been eliminated but is considered ,m]il~ely. The gaps associated with the R3 fragment, which is produced by the action of Hind and maps in the cistron A region on the t X genetic map, were found mostly in the viral strand; conversely all the 82p incorporated into gaps in the viral strand was found in the R3 fragment (Eisenberg & Denhardt, I974b). The 670 base-pair 1%3 fragment is cleaved by the Hae restriction endonuclease into four fragments (Lee & Sinsheimer, 1974). The Z6a (290 base-pairs) and the Z6b (285 base-pairs) fragments migrated under the conditions of electrophoresis of Figure 2(a) to the position of fractions 87 to 95. The two smaller fragments, containing 57 and 38 base-pairs, migrated to the position of fractions 35 to 45. Thus, in all the other fragments of Figure 2(a) the 32p label is presumably incorporated only in gaps associated with complementary strand DI~A. In the various fragments in Figure 2(a) that contain the 82p associated only with the complementary strand DNA, the 32P/all ratio varied significantly, in contrast to the 32P/3H ratio found for these fragments in Figure 2(b), suggesting that the gaps in the complementary strand were not randomly distributed on the ¢X genome. The absence of label at the top of the geI (right) indicates that the digestion has gone largely to completion. (b) Pyrimidine tracts Tre~ent in the gaTs of the c~mplementary strand The gap~ in ~bX R F II DNA isolated from ~bX-infected cells at a time of R F replication were completely filled in with [a-32P]dCTP and [a-32P]dTTP as described in Materials and Ylethods section. The complementary and viral strands of the [SH, 82P]RF II DNA, which were separated and purified as described, were subjected to pyrimidine oligonucleotide analysis. The pyrimidine tract pattern characteristic of the gaps present in the complementary strand of R F II DNA is shown in Plate I(c). Plate I(a) shows the pattern of pyrimidine tracts characteristic of the complete complementary strand which was synthesized in vitro as described in Materials and Methods, section (f). If the gaps in the complementary strand were randomly distributed over the ¢X genome, the pyrimidine tract patterns of the gap-labelled and complete complementary strands would be identical; clearly they are not. We note especially that the oligonucleotides CsT, CsTa, C2Tv, CsT4, CaTs and C~T7 were evident in the intact strand but were not clearly visible in the gap-labelled complementary strand DNA. In addition, the data in Table 1 show that the distribution of agp label among the pyrimidine tracts in the gap-labelled complementary DNA differed significantly from that of the complete strand. Thus, for example, while 2.0 and 1-4~o of the total 82p 8

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NON-RANDOM

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IN NASCENT

~X RF

115

TABLE 1

A c~omparison of the distribution of 82p among the ]pyrimidine tracts of gap-labeUed and comlalete strands Pyrimidine tract

V i r a l s t r a n d (%) Gap region Complete

Complementary strand (%) Gap region Complete

(1) T I CI

19.4 7.1

10.5 4-0

20.3 4-2

5.8 10-5

(2) T2 CT C2

12-5 14.8 4.3

6.7 13.8 4.2

16-2 15.4 3.1

5-5 14.5 8.9

(3) T s CT2 C2T Cs

3"9 11.8 5.4 0-9

3.4 10.9 4-9 0-2

3.6 10.1 5.5 0.6

2.1 6.7 9-6 1.1

(4) T , CTs C~Ta C3T C4

1.3 3.5 3.0 1.5 0.1

1-9 4.3 3.5 1.8 0.4

1.a 3.6 3.3 0.9 --

0.6 3.2 5.3 3.2 --

(5)

0-5 1.3 1"8 1"2 0-3

0.8 3.4 5"2 2"1 1.1

0.7 1-8 2"1 1"1 0"2

0-7 0"8 3"9 1"9 2.0

(6) T8 CT5 C2T4 CaT3 CaT2 CsT

-0.5 0-7 0-7 0-2 --

0.2 0.9 1.9 1-1 1"3 --

0-1 0-9 1.5 0.9 0"3 --

0.1 1-4 2-7 1.3 0.4 0"3

(7) CTe C2T5 C8T4 C4T8 CsT

0-1 0.2 0"2 0-1 2"5

0.5 1-3 1"7 1-1 0"6

0.3 0.6 0-5 0"3 --

0.3 1.2 0"7 0-4 --

(8) C2T8 C3T5 C4T4 C5T3

-0.1 0"1 --

0-7 0.7 0.7 0.4

0.3 -0.1 --

0.4 -1.4 0.4

-0-1

0"6 0-5

------

--0-6 0.4 0"3 0"3 0-2

--

0.5

---

---

0-6 0-5

T5 CT 4 C2T3

C3T2 C4T

(9) C2T7 C3Te C4Ta CsTa C6T3 (10) C2Ta CTTa (11) C3T8 C4T7

A c o m p a r i s o n o f t h e d i s t r i b u t i o n o f 82p label a m o n g t h e p y r i m i d i n e t r a c t s o f g a p - l a b e l l e d a n d complete strands of ~X RF DNA. The individual spots were located autoradiographically, scraped off t h e D E A E - e e l l u l o s e p l a t e s ( P l a t e s I a n d II), a n d t h e a m o u n t o f e~p a s s o c i a t e d w i t h e a c h in~vldual tract determined as described in Materials and Methods. In the Table the amount of 3~p l a b e l f o u n d i n e a c h t r a c t is e x p r e s s e d a s t h e p e r c e n t a g e o f t h e t o t a l r a d i o a c t i v i t y r e c o v e r e d f r o m t h e P l a t e . All t h e p y r i m i d i n e t r a c t s s e e n i n P l a t e s I a n d I I a r e listed. T h e p e r c e n t a g e rec o v e r e d i n e a c h o f t h e p y r i m i d i n e t r a c t s o f labelled D N A is r e p r o d u c i b l e w i t h i n 1 0 % .

116

S. E I S E N B E R G

ET

AL.

label recovered from the complete complementary strand was associated with C4T and C~T4, respectively, only 0-2~ and 0.1 ~ of the total 8~p label recovered from the gap-labelled DNA was associated with these tracts (Table 1). Al_so, a relative enrichment of 32p label in T1 and T~ over C1 and C2 in the gap-labelled strand was observed as compared to the intact strand. (c) Pyrimidine tracts pre~en~ in the gap of the viral strand Autoradiograms of the chromatograms of the pyrimidine oligonucleotides from the gap region of the viral strand of ~X R F II and of the oligonucleotides from uniformly 32p labelled single.stranded viral DNA are shown in Plate II. It is evident that the following ollgonueleotides of the viral strand are not clearly visible in the gap-labelled DNA: C~Ts, CsT4, C6T3, C7T8 and C2T8. A comparison of the relative intensities of labelling of the C6T, C2T3, C4T, C4T2, C8T4 and C4T4 tracts (shown in Plate II(c)) to the relative intensities of these pyrimidine tracts in the uniformly labelled viral DNA (Plate II(a)) indicates a marked enrichment of s2p label associated with C6T in the gap-labelled viral DNA strand. The absence of the C6T tract from the gap-labelled complementary strand of ~X R F H DNA (Plate I(e)) is an indication that the complementary and viral strands, which were fractionated on hydroxyapatite, were well separated from each other. The significantly different distribution of 82p among the pyrimidine tracts in the gap-labelled as compared to the complete viral strand was also verified when the radioactivity present in the individual tracts was quantitated (Table 1). (d) The average size and base.coml~osition of the gaps in the viral and complementary strands Determln~tion of the average size and base-composition of the gaps present in ~bX R F H DNA is complicated by the fact that the R F II DNA formed as a result of R ~ replication is not homogeneous in its structure. Within the population of RI~ II molecules isolated from ~X174-infected cells there are molecules with a single gap and molecules with multiple gaps (Eisenberg & Denhardt, 1974a). In order to estimate the size of the gaps, determination of the number of gaps present in the population of R F II molecules and the purity of the DNA preparation become crucial. The approach we used in these experiments, as described in Materials and l~Iethods, enabled an accurate estimation of the number of gaps and ensured that what we were looking at was only t X DNA. The femtomoles of 32p label incorporated into the in vitro formed R F I in different reactions with various radioactive substrates are presented in Table 2. In order to have an identical substrate for all the conversion reactions by T4 DNA polymerase and T4 ligase, the entire R F DNA preparation was first treated with bacterial alkaline phosphatase. R F II DNA that was treated with bacterial alkaline phosphatase could not be converted to R F I by DNA polymerase and ligase unless preineubated with polynueleotide kinase and ATP. The phosphatase-treated R F II was then reacted with polynucleotide kinase and ATP (8~p or 31p) to replace the terminal phosphates. From the amount of 82p incorporated in the reaction with [~-32P]ATP the number of 5' ends in the R F II could be deduced. Since none of the [82P]RF II could be converted to R F I by ligase alone, but some of it could when both ligase and T4 DNA polymerase were present in a gap-filling reaction, we believe that there were very few nicks present and that the

N O N - R A N D O M G A P L O C A T I O N I N N A S C E N T ~X R F

117

i n c o r p o r a t i o n of 82p gives a r e a s o n a b ] y a c c u r a t e m e a s u r e of t h e n u m b e r of gaps i n t h e R F I I p o p u l a t i o n . T h e absence of a significant a m o u n t of n i c k i n g is s u p p o r t e d b y t h e a s y m m e t r i c d i s t r i b u t i o n of 5' t e r m i n a l s2p b e t w e e n t h e v i r a l a n d c o m p l e m e n t a r y s t r a n d s (see T a b l e 3), a n d t h e fact t h a t i n our R F p r e p a r a t i o n s there was always a s u b s t a n t i a l a m o u n t of R F I. F r o m t h e a m o u n t of r a d i o a c t i v i t y i n c o r p o r a t e d i n t o R F I I (carrying 5' t e r m i n a l 81p) i n separate reactions w i t h each of t h e four [a-32P]dNTPs, T 4 D N A p o l y m e r a s e a n d p o l y n u c l e o t i d e ligase, t h e n u m b e r of each of t h e four deoxynucleotides could be d e t e r m i n e d . T h e p r o p o r t i o n o f t h e four d e o x y r i b o n u c l e o t i d e s G : A : C: T i n c o r p o r a t e d i n t o t h e i ~ vitro f o r m e d R F I was 1 : 1 - 3 : 1 - 5 : 2 . 3 , r e s p e c t i v e l y (Table 2). T h e r a t i o o f TXBLE 2

82p incawloorated into in vitro-formed R]~ I (fmot)

[7-s2P]ATP [a-32P]dCTP [a-32P]dTTP [~-~2P]dGTP [~-s2P]dATP

1

2

9 31 45 19 27

9 33 42 18 27

[3H]RF I I DNA was converted to RF I in five separate reactions, each with a different 32p. labelled triphosphate as described in Materials and Methods. Each reaction contained 6 × 104 to 103 SH ets/min of [3H]TdR-labellcd ~bX R F I I DNA. I n each reaction 20 to 25% of the total [3H]RF I t was converted to R F I. This percentage conversion is also obtained with R F I I DNA that was not exposed to bacterial alkaline phosphatase and polynucleotlde kinase prior to T4 DNA polymerase and T4 ligase treatment. The amount of s2p associated with R F I was normalized in all reactions to the same number of 3H counts (104 ets/min) and the fmol of incorporated 3~p was calculated from the specific activities of the different 32P-labelled triphosphates. Results obtained from two separate experiments with two different DNA preparations are presented. TABLE 3

Percentage of a~.p ,,corseted.out,, by unlabelled viral single-stranded DNA

[7-s2p]ATP [~-s2P]dCTP [m-s2P]dTTP [~-s2P]dGTP [a-s2P]dATP

1

2

29-8 30.2 32.2 30"9 31'7

38.7 39.9 33.9 33"6 38"0

The complementary and viral strands of the variously labelled R F I DNA preparations, which were obtained as described in the legend to Table 2, were separated and fractionated on hydroxyapatite columns as described in Materials and Methods. I n all reactions, 50 to 60% of the uniformly present 3H was displaced by the unlabelled viral DNA and eluted from hydroxyapatit~ in the low salt (0.14 M-potassium phosphate, pH 6.8) buffer. The percentage of the s2P-labelled DNA that was eluted with 0.14 M-potassium phosphate was corrected to 57% competition of the in v/reincorporated SH label (to correct for the larger number of thymine residues present in the viral strand than in the complementary strand). The recovery of SH and 8~p from the hydroxyapatite column~ was more than 90%. I n control experiments, annealkug without competition, more than 95% of the 8H and 32p label was eluted as double-stranded DNA. Results from the two experiments are presented.

118

S. E I S E N B E R G

ET

AL.

nucleotides incorporated into the molecules that were not converted to R F I was the same (data not shown), suggesting that the gaps in the R F I I that was converted in vitro to R F I were representative of the entire population of R F I I DNA. The amount of sap associated with the complementary and viral strands in each of the five reactions is shown in Table 3. From the data in Tables 2 and 3 the number of molecules of each of the four nucleotides incorporated into the gaps in the complementary and viral strands, on the average, can be calculated. These are given for the two independent experiments in Table 4. The average size of the gaps in the complementary and viral strands was 13 to 14 and 12 to 16 nucleotides, respectively. The average base-composition of the gaps in the complementary and viral strands differed from that of the complete strands as summarized in Table 5. The gaps in the complementary strands were enriched in T and deficient in A residues, whereas the gaps in the viral strand were enriched in C and deficient in G residues. TABLE 4

Average number of d N M P s incorporated per gap in CX R.F I I .DNA dCMP 1 2 Complementary s t r a n d Viral s t r a n d

3.5 3-7

3-6 3"7

dTMP 1 2 4.9 5"7

dGMP 1 2

5.1 4.0

2.1 2.4

2.2 1-7

dAMP 1 2 3-0 4.2

2-4 3-0

The a m o u n t of 3ap label associated w i t h viral a n d complementary strands was calculated from t h e dat~ contained in Table 2 a n d t h e percentage label t h a t was e l u t e d w i t h 0 . 1 4 ~ - p o t a s s i u m p h o s p h a t e (single-stranded viral DNA) a n d 0-3 m-potassium p h o s p h a t e (pH 6.8) solution (comp l e m e n t a r y strands in duplex DNA) (Table 3}. The average n u m b e r of t h e different deoxyribonueleotides incorporated into a single gap was calculated according to t h e e q u a t i o n described in Materials a n d Methods. Results obtained with two i n d e p e n d e n t D N A preparations are presented.

T~LE 5

Average base.composition of the gaps in CX R F I I D N A and the complete strands (°/o) Complementary s t r a n d Gaps Complete C T G A

26 37 16 20

24.1 24.6 18.5 32-7

Viral s t r a n d Gaps Complete 27 32 15 26

18.5 32.7 24-1 24-6

The base-composition of the average gap associated with t h e complementary a n d viral strands. This was calculated from t h e d a t a presented in Table 4; t h e two experiments h a v e been averaged. The base-composition of viral a n d complementary strands of ¢X174 D N A was determined b y Sinsheimer (1959).

4. Discussion (a) Comments on the strategy When the R F I I molecules were labelled in vitro in the gap-fil]ing reaction and then analysed using various means (restriction endonucleases, phosphatase-~ kinase, formic acid/diphenylamine hydrolysis) every effort was made to ensure that the various reactions had gone to completion. For example, if the T4 DNA polymerase

NON-RANDOM

GAP

LOCATION

IN NASCENT

SX RF

119

reaction was first carried out with unlabelled dNTPs (Materials and Methods, section (e) (iii)), and then subsequently with labelled dNTPs, no radioactivity was incorporated. Also, as shown in Figure 1, prior to, but not after, the gap-filllug reaction the RF I I molecules were sensitive to the single-strand specific nucleases from N. crassa. By these criteria all of the gaps in the R F I I molecules have been filled in. Control reactions with bacterial alkaline phosphatase, polynucleotide kinase, polynucleotide ligase and the restriction enzymes indicated that the reactions were not Hmited by lack of enzyme. In earlier work (Schekman eta/., 1971) it was observed that the conversion of RF I I to R F I in T4 DNA polymerase ~- ligase reactions was less than complete. The hypothesis was advanced at that time that those molecules that could not be converted to RF I had short tails, and some evidence was presented that such molecules existed, at least in TolA + cells. These molecules probably constitute the class of R F I I molecules observed by Razin & Sinsheimer (1970) to be resistant to the 5' to 3' exonuclease activity of DNA polymerase I (Masamune & Richardson, 1971). Another reason why not all of the RF II molecules are converted to RF I is that the hgase reaction may not have been 100% efficient. The conversion to R F I by ligase of RF II containing only one nick introduced by pDNase goes only to about 60~o completion (Schekman et al., 1971; Bartok & Denhardt, 1975). van de Sande et al. (1972) also noted that certain ligase reactions do not always go to completion. In the experiments reported here the RF II was isolated from a poIA strain and is thus less likely to have a significant proportion of "tailed" molecules, if such structures are the result of DNA polymerase I activity. We therefore assume that the molecules that were not converted to RF I were not converted because of the inefficient ligase reaction and not because of any difference in structure. In support of this assumption are the observations (data not shown) that the distribution among the restriction enzyme fragments and among the pyrimidine oligonucleotides of the sap incorporated into the gaps was'similar regardless of whether the RF I, the RF II, or a mixture was analysed. The reason we chose to analyse in detail only the RF II in the determination of the pyrimidine oligonucleotides of the separated strands (Results, sections (b) and (e)) was to avoid the artificial introduction of breaks into the R F I that would be necessary to separate the strands. The reason we chose to analyse the size of the gaps in only the RF I product (Results, section (d)) was because of a small and variable contamination with E. coli DNA fragments. Although this slight contamination of the RF I I with E. coli DNA did not interfere when the gaps were labelled with T4 DNA polymerase (presumably because the fragments were a poor substrate) they did make the quantitation of the ends with polynucleotide kinase suspect. This contamination was completely eliminated by isolating the R F I molecules either by sedimentation through alkaline sucrose gradients or centrifugation to equilibrium in CsC1 density gradients containing ethidium bromide. For the reasons mentioned above we believe that it is valid to extrapolate the results we have obtained on these separated components to the entire population of gap-containing R F I I molecules initially isolated from the cell. We cannot exclude the existence of some bias in our data however. For example that subset of nascent RF II molecules with only one gap in the viral strand may be converted to RF I more efficiently than that subset of nascent R F II molecules with multiple gaps in the complementary strand. This may explain why the total amount of label found in the viral strand gaps in the RF I (Table 3) is not less than 30%.

120

S. E I S E N B E R G

ET

AL.

(b) The gaps in the complementary strand T h e gaps that were found in nascent R F II DNA produced in vivo during R F rephcation appear in many places on the CX genome (Eisenberg & Denhardt, 1974a, b). In this report we have shown results obtained using three different techniques (the restriction enzyme analysis; the qualitative comparison of the distribution of pyrimidine tracts present in gaps associated with the complementary strand to the psuimidine tracts of the complete strand; and the average base-composition of the gaps found in the complementary strand DNA) that indicate a non-random distribution of the gaps on the CX genome. If the gaps are present as the result of an event occurring during the initiation of DNA synthesis, then their non-random location in the complementary strand of nascent RF II DNA suggests the existence of multiple and non-randomly distributed sites on the viral template at which synthesis of the complementary strand is initiated. I t is not clear yet what directs the initiation o£ DNA synthesis at these sites, nor do our data indicate whether all possible sites of initiation are used each time a complementary strand is synthesized. By the tests employed here we have not detected an enrichment for complementary strand gaps in any particular region of the genome. We do not have results that shed light on the mechanism of gap formation in the complementary strand during RF replication. The recent demonstration of a requirement for RIqA primer synthesis for the initiation of synthesis of the complementary strand in vitro (Schekman et al., 1972) and the requirement for the dnaG gene product, which may be a rifampicin-resistant RNA polymerase (Schekman et al., 1974), for in vivo RF replication (McFadden & Denhardt, 1974), is compatible with the idea that the gaps are sites where RNA primers were first synthesized and then excised after fulfilling their primer function.

(c) The gap in the viral strand Within the population of R F II molecules isolated from E. coli cells during R F replication there were molecules with gaps in the viral strand. There appeared to be only one gap located in the viral strand and it was restricted to the region of cistron A on the CX genetic map (Eisenberg & Denhardt, 1974b). The pyrimidine-tract analysis and the base-composition data presented above confirm the non-random location of the gap in the viral strand. The pyrimidine oligonucleotide analysis showed a striking enrichment o£ 32p label associated with C6T in the gap as compared to the complete strand, suggesting that CsT is part of the nucleotide sequence located in the region of the 5' end~ of the gap in the viral DNA. It is conceivable that this pyrimidine tract, which is not present in the complementary strand of CX DNA, is part of a specific signal for either termination or initiation of a round of rephcation. The great enrichment for label in Z6b relative to Z6a (fractions 87 to 95 in Fig. 2) suggests that the 5' end of tim gap, presumably containing the C6T tract, is in Z6b and that the 3' end ranges back into Z6a to a variable extent. We have estimated the average size of the gap in the viral strand to be 12 to 16 nucleotides long. This estimate is not easily reconciled with the large number of ~f The 5' end of the gap is the region adjacent to the 5' terminus of the strand containing the gap.

NON-RANDOM GAP LOCATION IN NASCENT SX RF

121

pyrimidine tracts found when the gap was filled in. Several explanations can be considered: (1) The size of the gap, when formed, is about 15 nucleotides, but it can be formed in many places within the cistron A region. (2) The mechanism for the formation of the gap in the viral strand during R F replication is such that a largo gap is formed (perhaps 200 to 300 nucleotides long), but that the gaps in most of the R F II molecules have been partially or completely closed in vivo. (3) There is no unique size for the gap formed in the viral strand. When formed, the gaps have various sizes. The majority of the molecules having a rather small gap, up to perhaps 50 nueleotides long. The enrichment of 32p associated with C6T suggests some sequence specificity for the gap in the viral strand and makes the first possibility less likely. We cannot distinguish between the latter two possibilities. The gaps in RF II formed during the period of viral single-stranded DNA synthesis were studied by Johnson & Sinsheimer (1974), and they found that there was one gap per RF molecule in the viral strand in the region of cistron A. However, two classes of RF II molecules were observed. The majority of the RF II molecules had a gap of about 100 nucleotides, but some 35~/o had a much smaller gap. The significance of these results, and how they relate to our results, is not clear. One possibility that our data appear to rule out is t h a t the gap in the nascent viral strand is at the site of one of the major hairpin loops in CX DNA. Bartok et al. (1975) have shown that the pyrimidine oligonucleotides characteristic of the hairpin loops resistant to the 2/. crassa single-strand specific nucleases include T4 and Ts. The data presented in this paper show no indication of an enrichment for these sequences in the gap in the viral strand. (e) Asymmetry in the initiation" and synthesis of viral and complementary strand D N A during R.F reTlication Baas & Jansz (1972) located the origin of R F replication in the cistron A region on the CX genetic map. Based on their genetic analyses they proposed that replication proceeds.from the cistron A region unidirectionally and clockwise on the CX genome. This has been confirmed recently by Godson (1974). Our results on the location and distribution of gaps in nascent RF II DNA produced as result of R F replication (Eisenberg & Denhardt, 1974a,b, and the results presented here) agree with the proposed origin and direction of replication; they suggest further than the origin of RF replication contains the CsT tract and is in the Hae Z6b cleavage fragment. The restricted location of the gap in the viral strand in the eistron A region and the fact that the gaps in the complementary strand can be formed at a moderate number of non-randomly located sites on the CX genome imply an asymmetry in the initiation and synthesis of the two strands. We imagine RF replication to occur as shown in Figure 3. Replication starts with the initiation of viral strand synthesis on an R F I molecule, at the origin of replication, in the region of cistron A. Synthesis proceeds continuously and clockwise on the genetic map, preceding the initiation of synthesis of the complementary strand (McFadden & Denhardt, 1975). The initiation of synthesis of the complementary strand occurs at non-randomly located sites on the displaced viral strand, perhaps because of a "start" signal or because a certain length of single-stranded DNA is

122

S. E I S E N B E R G

~T

AL.

Nick

RFI

Fie. 3. Model for ~X RF replication. Parental strands are represented by the thin lines. The thick lines represent newly synthesized DNA. Viral strand synthesis is initiated at the origin in the cistron A region, the synthesis proceeding continuously and in a eloekwise direction from the • origin of replication. The complementary strand DNA fragments are initiated at specific sites on the displaced parental strand. This meohanism of replication will give rise to a daughter molecule containing a single gap (g+) in the viral strand in the eistron A region, and a daughter molecule with multiple gaps (g-) at specific sites in the nascent complementary DNA. These daughter moleeules could enter the next round of replication after the gaps are filled in by the combined action of DNA polymerase I (usually) and DNA ligase. required. The synthesis of the complementary strand proceeds in the clockwise direction, discontinuously, b y multiple initiations at sites on the single-stranded viral template strand. This mechanism of replication results in the production of two daughter molecules with different structures. One daughter molecule contains the nascent viral strand with one gap in the eistron A region, probably with its 5' end somewhere in the Hae Z6b cleavage product. This is located at the site where the synthesis of the viral strand was initiated and terminated. The other daughter molecule contains multiple gaps present between the nascent complementary strand fragments. The gaps are normally closed b y DNA polymerase I (Eisenberg & Denhardt, 1974a) and sealed b y DNA ligase (McFadden & Denhardt, 1975). I n proposing this model we assume t h a t both strands, the viral and the complementary, are initiated b y an R N A primer, which is in contrast to the rollingcircle model proposed b y Gilbert & Dressler (1968) and Schr6der et al. (1973). Our assumption is supported by the fact t h a t the dnaG gene product, which was implicated in the synthesis of an R N A primer in vitro (Schekman e$ aL, 1974), is required in rive for the synthesis of the complementary and viral DNA during ~X174 replication (McFadden & Denhardt, 1974). An alternative possibility is that the gaps represent the site of action of a protein (9 protein) that interacts directly with the DNA template to allow DNA polymerase to initiate synthesis (Denhardt, 1972).

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