phage G4oric complex required for primer RNA synthesis1

J. Mol. Biol. (1998) 276, 689±703

Structure of the Escherichia coli primase/Singlestrand DNA-binding Protein/Phage G4oric Complex Required for Primer RNA Synthesis Wuliang Sun and G. Nigel Godson* Biochemistry Department New York University Medical Center, 550 First Avenue New York, NY 10016, USA

Escherichia coli primase/SSB/single-stranded phage G4oric is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small singlestranded G4oric fragments, we have identi®ed the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have de®ned the structure of the primase/SSB/G4oric priming complex. Two SSB tetramers bind to the G4oric secondary structure, which dictates the spacing of 30 and 50 bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to speci®c DNA sequences in the 30 and 50 SSB-free G4oric regions. Binding of the 30 SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template. # 1998 Academic Press Limited

*Corresponding author

Keywords: primase; SSB; G4oric; stem-loop structure; antisense oligonucleotide

Introduction Primases are an essential component of the machinery of DNA replication initiation in both prokaryotes and eukaryotes. They function to synthesize short primer RNA (pRNA) on DNA templates, that is then used for DNA strand elongation by DNA polymerases. To study Escherichia coli primase (referred to as primase in this paper), there are two major in vitro priming systems: primosome and G4. In the primosome system, primase is part of a multiprotein mobile complex and synthesizes pRNA at numerous sites on template DNA (Kornberg & Baker, 1992; Marians, 1992). The pRNA initiation sites do not occur at random, usually starting at 50 CTG 30 sequences (Yoda & Okazaki, 1991). Based on the different initiator proAbbreviations used: BSA, bovine serum albumin; dsDNA, double-stranded DNA; nt, nucleotide(s); RF, replicative form; ssDNA, single-stranded DNA; SSB, single-strand DNA-binding protein; PCR, polymerase chain reaction. 0022±2836/98/090689±15 $25.00/0/mb971471

tein, primosomes can be further classi®ed into two subtypes: PriA-dependant (such as single-stranded phage fX174 (Arai & Kornberg, 1981; Ng & Marians, 1996) and plasmid ColE1 (Nomura et al., 1982)) and DnaA-dependant (exempli®ed by E. coli chromosome (Kaguni & Kornberg, 1984; Messer & Weigel, 1996)). Single-strand DNA-binding protein (SSB) is required in all primosome priming systems (Meyer & Laine, 1990). In the single-stranded phage G4 system, primase synthesizing pRNA requires only SSB and starts at a unique 50 CTG 30 sequence in the origin of complementary DNA strand synthesis (G4oric). The G4 type of priming also occurs in related single-stranded phages a3, St-1 and fK (Sims et al., 1979) and some plasmids such as R1 (Masai & Arai, 1989). Because of its simplicity and single pRNA initiation site, the G4 priming system provides an ideally simple model to study how primase interacts with its recognition DNA sequence on SSB coated templates. In phage G4 and related phages, the oric contains three stem-loops (de®ned as stem-loop I, II and III) and pRNA synthesis is initiated at the thymidine # 1998 Academic Press Limited

690 residue of the 50 CTG 30 trinucleotide sequence, located four nucleotides (nt) 30 from the base of stem-loop I (Fiddes et al., 1978; Sims & Dressler, 1978). An in vivo study indicated that the smallest functional G4oric sequence is 140 nt long, consisting of a 100 nt core stem-loop structure with 25 nt on the 50 ¯ank and 15 nt on the 30 ¯ank (Lambert et al., 1986). Mutagenic analysis demonstrated that there were different signals in G4oric required for conversion of the viral single-stranded DNA (ssDNA) to the duplex replicative form DNA in vivo, including the distance between the 50 CTG 30 sequence and the base of stem-loop I, the secondary structure of the stem-loop I and the sequence of stem-loop III (Hiasa et al., 1989, 1990). A nuclease footprinting study of primase interaction with the G4-like phage fKoric showed that primase protection appeared on several regions consisting of stem-loops I and III (Sims & Benz, 1980). A stoichiometric study using gel ®ltration reported that two primase molecules bound on one G4oric which was cloned into a viral ssDNA (Stayton & Kornberg, 1983). These early experiments were mostly carried out on G4oric cloned in circular ssDNA vectors. Because of the large size of the viral DNA, it was dif®cult to study the G4oric structure and participation of SSB in initiation of pRNA synthesis. To avoid these problems, we have adopted a G4 priming system using small G4oric ssDNA fragments, so that we can use gel retardation, footprinting and pRNA synthesis to study in detail the structure of the priming complex. Using this system, we have shown that two SSB tetramers bind to the G4oric stem-loop structure in a ®xed position, leaving part of stem-loop I and the 50 CTG 30 initiation site as SSB-free DNA (Sun & Godson, 1993). In this paper, by using a strategy of deletion and of speci®cally and locally blocking protein binding sites through annealing antisense oligonucleotides to G4oric ssDNA, we show that: (1) in addition to the sequence 30 of the stem-loop structure that contains the 50 CTG 30 pRNA initiation site (Hiasa et al., 1989), the sequence immediately 50 of the stemloop structure is also required for primase to function; (2) besides the two SSB tetramers bound to the stem-loop structure (Sun & Godson, 1993), binding of a third SSB tetramer upstream of the initiation site on the region 30 of the hairpin structure is necessary for pRNA synthesis; binding a fourth SSB tetramer on the 50 side, although not essential, increases pRNA synthesis to wild-type level; and (3) two primase molecules bind on one G4oric (Stayton & Kornberg, 1983), one on each side of the secondary structure in the SSB-free regions between the stem-loop bound SSB and the 30 and 50 adjacent SSB tetramers. From these data, we de®ne the structure of the primase/SSB/G4oric priming complex in which two SSB tetramers bind to the G4oric secondary structure to dictate the spacing of adjacent 30 and 50 bound SSB tetramers and two primase molecules then bind separately to speci®c G4oric sequences in 30 and 50 SSB-free

Structure of Primase/SSB/G4oric Priming Complex

regions. The general signi®cance of this mechanism of speci®cally exposing DNA-binding sites to primase or initiator proteins by phasing of SSB at a stem-loop structure is discussed. Moreover, we present here some novel techniques including antisense oligonucleotide protection and pRNA synthesis on an SSB-melted, double-stranded DNA (dsDNA) template.

Results 50 deletion G4oric: requirement of the short 50 region flanking stem-loop III for primase function A 278 nt G4oric ssDNA fragment containing the 100 nt core stem-loop structure plus 100 nt of 50 and 78 nt of 30 ¯anking sequences can be used by primase to synthesize pRNA with the same ef®ciency as the viral ssDNA containing G4oric (Sun & Godson, 1996) which is referred as the wild-type G4oric fragment in this paper. It is known that primase does not synthesize pRNA on naked G4oric ssDNA, but requires SSB (Zechel et al., 1975), and the structure of the SSB/wild-type G4oric fragment complex has been de®ned as a formation of four SSB tetramers binding to the DNA with two on the stem-loops, one on the 50 side and one on the 30 side (Sun & Godson, 1993). In the following experiments, when we used deletions of G4oric to study the interaction of primase with G4oric, we always analyzed the structure of SSB binding ®rst. To investigate whether the sequence 50 to the base of stem-loop III of G4oric is necessary for primase interaction, we used a pair of G4oric ssDNA fragments, G4oric 333 and 310, that differed only in the length of their 50 ¯anking sequence (see Figure 1A). Both fragments contained the same 100 nt core stem-loop structure and 210 nt ¯anking the 30 side of stem-loop I, but G4oric 333 contained 23 nt 50 ¯anking the base of stem-loop III and G4oric 310 had none. When the number of SSB tetramers binding to these G4oric fragments was measured by SSB titration and gel retardation, both DNA were bound by four tetramers at saturation (Figure 1B, lanes 4 and 5 in both gels). From the previous study (Sun & Godson, 1993) of SSB binding to different sized G4oric fragments, we know that in these SSB/G4oric complexes two SSB tetramers would be bound to the G4oric stem-loop structure and another two bound on the 210 nt 30 tail. When primase was added to the SSB saturated complexes, the G4oric 333/SSB complex gave a further gel shift (Figure 1B left, lane 6); adding an unrelated protein bovine serum albumin (BSA), did not result in a further gel shift (lane 7), which indicated a stable primase binding. By contrast, the G4oric 310/SSB complex, like BSA, did not give a reproducible shift with primase (Figure 1B right, lanes 6 and 7), even at higher primase concentrations (data not shown). These results suggested that the 23 nt

Structure of Primase/SSB/G4oric Priming Complex

691 50 of stem-loop III was not bound by SSB but was involved in the stability of primase binding. In DNase I footprinting experiments, an identical pattern of cleavage was induced by SSB binding to the stem-loop structure on both G4oric 333 and G4oric 310 (Figure 1C shows only footprinting on the stem-loop I region). This was the same as observed from the wild-type G4oric fragment (Sun & Godson, 1996). Adding primase to the 333 and 310 nt G4oric/SSB complexes resulted in protection of the DNase I cleavage around stem-loop I on both G4oric DNA (Figure 1C). Similar results were obtained from micrococcal nuclease footprinting of primase interacting with two G4oric/SSB complexes (data not shown). The nuclease footprinting data suggested that, when the short 50 sequence ¯anking stem-loop III was deleted in G4oric, there was still primase interaction around stem-loop I. The template activities of these two G4oric/SSB complexes were then examined by pRNA synthesis assay. Since it is not easy to obtain a relatively large amount of G4oric ssDNA required for pRNA synthesis in vitro, we developed a new pRNA synthesis method that can use small dsDNA G4oric fragments generated by polymerase chain reaction (PCR). The dsDNA fragments were heat denatured and slowly cooled in the presence of SSB (SSB is heat stable) prior to synthesis reaction. pRNA synthesized on the SSB = melted G4oric dsDNA templates was full length, and the ef®ciency was approximately one third of that from the same sequence of ssDNA template. On the G4oric 333/SSB complex, primase synthesized normal length (25 to 29 nt) pRNA (Figure 1D, left part). The relative ef®ciency from 333 G4oric dsDNA was 27% compared with the ssDNA control of circular R199/G4 template (0.1 and 0.03 pmol were used, respectively), which was equal to approximately 70% to 80% of dsDNA con-

Figure 1. Deletion of the short 50 ¯anking sequence: comparison between G4oric 333 and G4oric 310. A, Structure of G4oric 333 and G4oric 310. The basic structure of G4oric is taken from Fiddes et al. (1978) and Lambert et al. (1986). The 50 CTG 30 , the pRNA initiation site, is marked. The nucleotide lengths are given and the regions binding SSB tetramers are indicated (see the text). B, Gel retardation assay of SSB binding and primase interaction. G4oric 333 and 310 ssDNA fragments (approximately 0.1 pmol) were (50 -32P) end-labeled. Concentration of SSB tetramer increased from 0.09 to 0.9 pmol and primase or BSA (as a negative control)

was 3.8 pmol. 8 mM MgCl2 was added in the electrophoresis system so that the Mg2‡ concentration was as the same as that in binding reaction. C, DNase I footprinting of the binding of SSB and primase on G4oric 333 and 310 ssDNA. G4oric ssDNA was (50 -32P) endlabeled. As shown here are the footprinting on stemloop I region of G4oric. The size marker was the ddA and ddC sequence of wild-type G4oric fragment. D, pRNA synthesized by primase on SSB = melted dsDNA fragments of G4oric 333 and 310. Left, the synthesis products from standard condition with four rNTPs and [a-32P]GTP. Right, pRNA synthesized under limited condition with ATP, TTP, GTP, [a-32P]GTP and ddCTP. The amounts of DNA templates used were 0.03 pmol ssDNA of circular R199/G4oric, 0.1 pmol dsG4oric 333 in both experiments, 0.23 pmol dsG4oric 310 in the four rNTP reaction and 0.45 pmol in the three rNTP reaction. The size marker was (50 -32P)-labeled oligonucleotides. The ef®ciency of pRNA synthesis (expressed as fmol [a-32P]GMP incorporated into synthesized pRNA per pmol dsDNA template) and the relative ratios are shown beneath each lane.

692

Structure of Primase/SSB/G4oric Priming Complex

Figure 2. pRNA synthesis on a series of 50 deletion G4oric. A, Structure of the serial 50 deletions. Fourteen serial deletions are shown the 50 side of stem-loop III region, together with wild-type G4oric 278 nt fragment. The 50 deletion fragments that were intensively analyzed are framed. The double-ended arrows indicate the three stem-loops. 50 CTG 30 is the pRNA initiation site. B, pRNA synthesis on the series of 50 deletion G4oric using SSB = melted dsDNA. Each dsG4oric (0.12 pmol) and ssR199/G4oric (0.06 pmol) were used in this experiment. Arrows indicate the same G4oric fragments assayed in both the left and the right gels. C, Relationships between the 50 ¯anking sequence and pRNA synthesis ef®ciency. The ef®ciency of pRNA synthesis plotted against the 50 end sequences of the series of 50 deletion G4oric. Broken line indicates continuous deletions from G4oric 333 to 328 (50 ) containing single nucleotide deletion and continuous line indicates non-continuous deletions from G4oric 326 to 294 containing several nucleotides deletion. Some deletions whose activity changed obviously are marked. The underline indicates the 50 part of stem-loop III.

trol (see the paragraph above and Figure 2, below). The synthesis ef®ciency, calculated from incorporation of [a-32P]GMP in the product, was 0.4 pRNA molecule per G4oric 333 dsDNA template (see Materials and Methods). On the 310 G4oric dsDNA/SSB template, however, the relative synthesis ef®ciency was only 1% (i.e. 0.02 pRNA per template) of ssDNA control and the pRNA synthesized was smaller in size (18 to 20 nt). To determine whether the small pRNA species started at the T of the 50 CTG 30 in template sequence 50


GTCCCTACTG


30 , cytidine 50 -triphosphate (CTP) was replaced by 20 , 30 -dideoxycytidine 50 -triphosphate (ddCTP) in the reaction. Because primase can use a mixture of ribo- and deoxyribo-

NTPs for pRNA synthesis (Rowen & Kornberg, 1978), use of ddCTP should give rise to a 9 nt product (i.e. termination at the ®rst G residue on template). As shown in Figure 1D (right part), both the 310 and 333 G4oric/SSB templates generated a single 9 nt product, the same as the control R199/ G4oric/SSB template. The relative ef®ciencies were similar with that observed under normal conditions. The de®cient synthesis and lack of primaseinduced gel shift on the 310 nt G4oric/SSB suggested that the short 50 sequence ¯anking stem-loop III is involved in stability of primase binding and primase processivity during pRNA synthesis.

Structure of Primase/SSB/G4oric Priming Complex

Serial 50 deletions: effect of removing the 50 flanking nucleotides on pRNA synthesis In order to de®ne in more detail the sequence 50 to the base of stem-loop III that is necessary for normal pRNA synthesis, a series of deleted G4oric fragments were made from the 50 end of the active G4oric 333 (see Figure 2A). The deletions began in the 23 nt of 50 ssDNA region, preceding stem-loop III (G4oric 333 to 310) into the 50 side of the stem (G4oric 306 to 294). The sequence between the 50 ends of G4oric 313 and G4oric 294 is a repeated sequence that also occurs at the 30 side of stem-loop I (Sims & Dressler, 1978). All these G4oric fragments were tested for pRNA synthesis activity using the method of SSB = melted dsDNA. Figure 2B shows the pRNA synthesized on these templates and Figure 2C is a plot of the synthesis activities that were calculated from two to ®ve independent experiments. As shown in Figure 2, the pRNA synthesized from the templates decreased in both amount and size as the deletion extended from G4oric 333 (containing 23 nt of 50 tail) to 319 (containing 9 nt of 50 tail) (Figure 2B, lanes 4 to 12). For example, the average amount of pRNA synthesized from the G4oric 333 template (lanes 4 and 40 ) was 73% compared with wild-type G4oric fragment (lane 3) and decreased to 18% on the G4oric 319 template (lanes 12 and 120 ). Within this region, a sharp reduction of pRNA synthesis occurred between G4oric 331 (lane 6) and G4oric 330 (lane 7) that contained 21 and 20 nt 50 ¯anking sequence, respectively. However, a further deletion that left only three adenine residues ¯anking the 50 side of stem-loop III (G4oric 313, lane 17), unexpectedly restored the template activity to approximately 47% that of wild-type G4oric. Removing these three adenine residues (G4oric 310, lanes 13 and 130 ) again reduced pRNA synthesis activity to a basal level (4% of that of the control). The furthest 50 deletions (G4oric 306, 301 and 294, lanes 14 to 16) had the same low level of template activity. Thus, 20 to 21 50 ¯anking nucleotides, especially the three adenine residues, are essential for the activity and processivity of primase during pRNA synthesis on SSB bound G4oric. Sequence and structure of the 30 flank of stemloop I required for pRNA synthesis: the 50 CTG 30 is not sufficient and binding of an 30 SSB tetramer is required To investigate whether the short sequence ¯anking stem-loop I of G4oric that contains the pRNA initiation site provides enough information on the 30 side for primase to interact stably and synthesize pRNA, another pair of G4oric fragments, G4oric 354 and 328, were designed and examined. Besides the same regions of the 100 nt core stem-loop structure and 228 nt 50 ¯anking stem-loop III, G4oric 354 contained 26 nt on the 30 side of stem-loop I (including

693 the initiation site) and G4oric 328 had no 30 ¯anking nucleotides. As the 30 short ¯ank region remains SSB-free (Sun & Godson, 1993), this pair of G4oric should form similar SSB/G4oric structures with two SSB tetramers bound to the hairpins and two bound to the 50 ¯anking region. The G4oric 328/SSB complex that had no pRNA initiation sequence lost almost all interaction with primase in gel shift and DNase I footprinting experiments (not shown) and had no pRNA synthesis activity (Figure 3A, lane 2). The G4oric 354/ SSB complex that contained the initiation site induced a typical primase protection pattern on the stem-loop I region in DNase I footprinting (not shown). However, this complex could not be consistently bound by primase in a gel retardation experiment (not shown). Moreover, its pRNA synthesis activity was very low, only 5% from the SSB-melted dsDNA and 15% from ssDNA compared with ssDNA control R199/G4oric (Figure 3A, lanes 4 to 6). The size of pRNA synthesized from G4oric 354/SSB, contrary to the small pRNA synthesized from G4oric 310/SSB (lane 3), was normal. These results indicated that on the 30 side of stemloop I, the 50 CTG 30 sequences are not enough for normal primase function. Some additional information must be required. A clue to this extra information was suggested from the structure of the wild-type G4oric fragment/SSB complex that contained four SSB tetramers with two bound to the stem-loops structure and one on the 50 and 30 ¯anks, respectively (Sun & Godson, 1993). We have already shown that binding of an SSB tetramer on the 50 ¯ank is not necessary for primase activity (see above). The instability of primase binding and the severe reduction of pRNA synthesis observed from G4oric 354 (it has only 26 nt 30 of stem-loop I) may be due to the absence of binding of a 30 SSB tetramer in the SSB/G4oric complex. To test this possibility, G4oric 256 was designed (Figure 3B). Besides the 100 nt stem-loop region and an essential 23 nt 50 ¯anking sequence, G4oric 256 contained 133 nt ¯anking the 30 side of stem-loop I. Gel retardation experiments showed that G4oric 256 was bound by three SSB tetramers, suggesting two SSB tetramers bound on the hairpin region and one on the 30 ¯ank. Two controls, G4oric 149 and G4oric 244, were assayed simultaneously. G4oric 149 contained the 100 nt core region and the same 23 nt on the 50 ¯ank but only 26 nt on the 30 ¯ank; it was bound by two SSB tetramers (Figure 3C). G4oric 244 contained an extended 118 nt 50 ¯ank and was bound by three SSB tetramers. The third SSB tetramer must therefore have bound on the extended 50 side of G4oric 244 (Figure 3D). When primase was added to these SSB/G4oric complexes in the same gel retardation experiments, only the SSB/G4oric 256 that contained the long 30 tail induced a further shift. However, this primase gel shift was an incomplete, partial one. (It was found in our experiments that four SSB tetramers are required by primase for a stable and complete

694

Structure of Primase/SSB/G4oric Priming Complex

Figure 3. The 30 ¯ank of G4oric: pRNA synthesis from G4oric 354 and G4oric 328; effect of binding of the third SSB tetramer on pRNA synthesis from G4oric 256, 149 and 244. A, pRNA synthesis on SSB/ G4oric 328 and SSB/G4oric 354. The amounts used were 0.12 pmol for each ds or ssG4oric fragments and 0.06 pmol of ssR199/G4oric circle. The ef®ciency and relative ratios are shown below lanes. B to D, Structure of G4oric 256, G4oric 149 and G4oric 244 and their interaction with SSB and primase assayed by gel retardation. The increasing concentration of SSB tetramer used in gel shift were 0.09 to 0.9 pmol for both G4oric 256 and G4oric 244 and 0.05 to 0.65 pmol for G4oric 149. Primase or BSA was 3.8 pmol. E, pRNA synthesis assay on SSB/ G4oric 256, SSB/G4oric 149 and SSB/G4oric 244 using SSB = melted dsDNA. The amounts of DNA used were as the same as that in A. The relative ef®ciency was measured by scanning the density of the pRNA bands from X-ray ®lms. Each panel represents the average calculated from at least four experiments.

gel shift, as shown in Figure 1B, left panel.) The SSB/G4oric 149 complex did not give a further shift with primase, nor did the SSB/G4oric 244 complex (Figure 3B to D). When the interaction of primase with these SSB/G4oric complexes was analyzed by DNase I footprinting, a typical primase protection pattern from nuclease cleavage on stemloop I region occurred only with the SSB/G4oric 256 complex (not shown). The pRNA synthesis activities of these G4oric/ SSB complexes were assayed using SSB = melted dsDNA (Figure 3E). SSB/G4oric 256 had most activity (26% compared with ssR199/G4oric, i.e. 70 to 80% activity if compared with dsDNA control) and generated normal sized pRNA. The SSB/

G4oric 149 complex had nearly no activity (<1%), as did the negative control R199 viral DNA that does not contain a G4oric sequence. The SSB/G4oric 244 had low activity (4% of that from ssR199/ G4oric) like the SSB/G4oric 354. The far 30 ¯anking sequences (a gene-coding region) probably do not contain speci®c sequence for primase binding because there is no sequence homology after 27 nt from the base of stem-loop I among oriC of phages a3, St-1 and fK (Sims & Dressler, 1978). We have previous shown that the 50 CTG 30 region is SSB-free. These data therefore suggested that binding of an SSB tetramer on the 30 side (i.e. upstream) of the pRNA initiation

Structure of Primase/SSB/G4oric Priming Complex

sequence on the 30 ¯ank of G4oric is required for primase activity. Complementary oligonucleotides block SSB binding: confirmation of the requirement of binding an SSB tetramer upstream of the 50 CTG 30 To con®rm that primase requires an SSB tetramer bound upstream of the pRNA initiation site, a novel technique was adopted that used antisense oligonucleotides to selectively block SSB binding on the 30 ¯ank region of the active G4oric 256. Five small oligonucleotides of 15, 20, 20, 26 and 26 nt were hybridized to the 30 107 nt of G4oric 256, which left the 26 nt adjacent to the base of stemloop I as unbound ssDNA (see Figure 4A). The resulting partial dsG4oric 256 therefore contained the same ssDNA region as the inactive G4oric 149 ssDNA. We used several small oligonucleotides instead of a single large one in order to eliminate an SSB-binding competition between the ssDNA template and the complementary oligonucleotide. To test whether the oligonucleotides could bind to G4oric, one to ®ve oligonucleotide(s) were annealed to G4oric 256, and the partially dsG4oric 256 fragments were mobilized in a native polyacrylamide gel. As the number of bound oligonucleotides increased, migration of the G4oric fragments decreased (Figure 4B), indicating stable and complete annealing. When the number of SSB tetramers bound to the partial dsG4oric 256 with ®ve annealed oligonucleotides was assayed by gel retardation, only two SSB tetramers bound, contrary to three SSB tetramers binding on the unannealed G4oric 256 ssDNA (Figure 4C). This result demonstrated that an SSB tetramer did bind on the 30 distant ¯ank region in the SSB/G4oric 256 ssDNA complex. When the template activity of the ®ve oligonucleotides blocked G4oric 256/SSB complex was assayed by pRNA synthesis, the amount of pRNA decreased to the level similar to that from the SSB/G4oric 149 complex (Figure 4D). These results con®rmed the conclusion obtained above that for normal pRNA synthesis activity, primase requires an SSB tetramer bound upstream of the pRNA initiation site on the far 30 ¯anking region of G4oric. Gel filtration: one primase molecule binds to the 50 flank and another primase molecule binds to the 30 flank of G4oric The above deletion study together with the previous nuclease footprinting data (Sims & Benz, 1980; Sun & Godson, 1996) suggested that the 50 and 30 ¯anking regions adjacent to the stem-loop structure of G4oric are directly involved in primase binding. An early stoichiometric study using gel ®ltration reported that two primase molecules bound to one G4oric that was cloned in R199 circular ssDNA (Stayton & Kornberg, 1983). Do both molecules of primase bind to the 30 ¯anking region

695 containing the 50 CTG 30 sequence or does one bind there and the other bind on the 50 ¯ank? To answer this question, we utilized two antisense oligonucleotides (both were 23 nt in length and were unable to be bound by SSB) to block either one or both of the 50 and 30 sequences ¯anking the base of the stem-loop structure in G4oric ssDNA (see Figure 5, top). We then measured the binding number of primase molecules to the SSB/G4oric/ oligonucleotide complexes by gel ®ltration. In order to estimate accurately the amount of G4oric ssDNA fragment (which is critical for the stoichiometric study), we used 32P uniformly labeled G4oric 302 nt ssDNA whose quantity could be calculated from the [a-32P]AMP incorporation. This concentration of [32P]G4oric was then veri®ed by using it to get a correct stoichiometry of [3H]SSB binding through gel ®ltration (In a previous study (Sun & Godson, 1993), we had determined the binding number of SSB tetramers on the G4oric 278 fragment). Primase was labeled with 3H in vivo and its concentration and speci®c activity were measured. As the speci®c activity of the [32P]G4oric 302 was higher than that of [3H]-primase (3  106 cpm/pmol and 1.7  103 cpm/pmol, respectively), cold G4oric 278 nt ssDNA was added into binding reaction so that the 32P and 3H counts in the primase/SSB/G4oric complex could be comparable. Firstly, we measured the number of primase molecules binding on the intact G4oric ssDNA/SSB complex with no antisense oligonucleotides annealed. The 32P and 3H-labeled primase/SSB/ G4oric complex eluted in the void volume prior to the excess of unbound [3H]primase (see Figure 5A). The molar ratio of primase to G4oric ssDNA in the 3 H/32P peak fraction was calculated as 1.9 (see Table 1, experiment A). (It should noted that the data in Figure 5A to D were normalized so that the results of these experiments could be compared; the original data from peak fractions are given in Table 1). To con®rm this result, the speci®c activity of the [32P]G4oric ssDNA was changed by adding twofold more cold G4oric 278 ssDNA in reaction. A similar molar ratio (1.7) of primase to G4oric in the 3H/32P peak fraction was obtained (detailed data not shown). This stoichiometry of primase binding using a small G4oric ssDNA fragment was consistent with Stayton & Kornberg's (1983) report using cloned R199/G4oric viral ssDNA circle. Antisense oligonucleotides were then annealed on the G4oric ssDNA to block the 50 and 30 ¯anking sequences, and the primase binding number measured by gel ®ltration. The ratio of oligonucleotide to G4oric was 14:1 and ratios of SSB to G4oric were 21:1 or 28:1 (with one or two oligonucleotides, respectively). These ratios were established as those giving the best inhibition of template activity (tested by pRNA synthesis) while allowing complete binding of SSB (measured by gel retardation) to G4oric ssDNA. When only the 50 antisense oligonucleotide was added to block the sequence 50 of stem-loop III, the counts of [3H]pri-

696

Structure of Primase/SSB/G4oric Priming Complex

Figure 4. Changing SSB binding and pRNA synthesis of G4oric 256 with complementary oligonucleotides. A, A diagram of G4oric 256 ssDNA (plus strand) and the locations of the ®ve (I to V) complementary oligonucleotides (minus strand) with indicated lengths. B, Annealing of oligonucleotides on G4oric 256 ssDNA assayed by gel electrophoresis. 0.2 pmol 32P-labeled G4oric 256 was incubated with different numbers of oligonucleotides (0.3 pmol each) and samples were mobilized in a non-denaturing 4% polyacrylamide gel. Arrows with dashed line show which oligonucleotide(s) was/were used in each lane. C, The number of SSB binding measured by gel retardation. Left, on the original ssG4oric 256. Right, on the ®ve oligonucleotides blocked G4oric 256. The amounts of components used were 0.2 pmol [32P]G4oric 256, 1 pmol each oligonucleotides, 0.17 to 1.4 pmol SSB tetramer in the left gel and 1.4 to 11.2 pmol SSB tetramer in the right gel. Electrophoresis was carried out at 4 C. Arrows with a broken line above indicate which G4oric were used in gel shift. D, pRNA synthesis from the partial ds-G4oric 256/SSB complex. These reactions contained 0.6 pmol ssG4oric 256, 8.5 pmol each of ®ve oligonucleotides, 8.5 pmol SSB tetramer, 11 pmol primase, four rNTPs and 10 mCi each [a-32P]GTP, [a-32P]CTP and [a-32P]UTP. The reaction mixture was also checked by gel retardation to make sure that the template was saturated with SSB (not shown). The arrows with a broken line above show which template was used in each synthesis reaction. R199 and R199/G4oric circular ssDNA were used as negative and positive controls. The size marker was (50 -32P)-labeled oligonucleotides.

mase in the 32P/3H peaks fraction dropped to just half of that on the intact G4oric; the molar ratio of primase binding on G4oric ssDNA correspondingly decreased to 1.0 (experiment B in both Figure 5 and Table 1). Experiments using another slightly

longer 50 antisense oligonucleotide (28 nt in length with ®ve extra 30 nt, but with the 50 end still terminating-ending at the base of stem-loop III) to block the 50 ¯anking sequence also gave a 1.0 molar ratio of primase to G4oric (data not shown). When both

697

Structure of Primase/SSB/G4oric Priming Complex

with different preparation of [32P]G4oric ssDNA and similar stoichiometric results were obtained (data not shown). The gel ®ltration results demonstrated that the immediate sequences ¯anking the 50 and 30 sides of stem-loops in G4oric contain primase binding DNA sequences; each of the sequences is bound separately by one primase molecule and two molecules of primase bind to G4oric in the primase/SSB/ G4oric complex. This interpretation may explain the low pRNA synthesis activity of G4oric 310 (Figure 1D) that completely deletes the 5 ¯anking sequence and absence of activity of G4oric 328 (Figure 3A) that completely deletes the 30 ¯anking sequence.

Discussion Structure of the primase/SSB/G4oric priming complex

Figure 5. Gel ®ltration assay of binding of [3H]primase to [32P]G4oric blocked by antisense oligonucleotides. Top, DNA sequence of the 50 and 30 regions ¯anking the secondary structure of G4oric (Fiddes et al., 1978) and location of the antisense oligonucleotides. A ± D, Gel ®ltration was carried on through 5 ml Bio-Gel A-0.5 m agarose gel column, and 90 ml fractions were collected (see Materials and Methods). For purposes of comparison, the 32P and 3H counts in some of the plots have been adjusted to equalize the pmols of [32P]G4oric in the original binding reaction and to allow for radioactive decay; the unadjusted 32P and 3H counts in peak fractions are given in Table 1. Structures of G4oric ssDNA and the annealed antisense oligonucleotide(s) are illustrated above each chart. The DNA templates used in each experiment are A, G4oric ssDNA alone, B, G4oric annealed with the 50 oligonucleotide, C, G4oric annealed with the 50 and 30 oligonucleotides and D, G4oric annealed with the 30 oligonucleotide.

of the 50 and the 30 sequences ¯anking the secondary structure of G4oric were blocked by adding two antisense oligonucleotides together, no 3H cpm was detected in the 32P peak fraction (experiment C), indicating that no primase molecules bound to the complex. When only the 30 antisense oligonucleotide was used to block the sequence on the 30 side of stem-loop I including the pRNA initiation site, surprisingly, the 3H cpm in the proteins-DNA binding fraction reappeared and the ratio of primase to G4oric was 1.1 (experiment D). This suggested that even when the pRNA initiation site was blocked by the antisense oligonucleotide, one primase molecule could still bind to the SSB/ G4oric complex on the 50 side. The antisense oligonucleotide protection experiments were repeated

Suf®cient experimental data have now been accumulated in this and other studies to depict the gross structure of the primase/SSB/G4oric binding complex during pRNA synthesis (Figure 6). The data to be accounted for are the following. (1) Two SSB tetramers (SSB tetramers A and B in Figure 6) bind on the secondary structure in G4oric ssDNA, probably forming an octamer (Sun & Godson, 1993). Hirao et al. (1990) reported that a sequence 50 GCGAAAGC 30 in stem-loop I potentially formed an extremely stable hairpin structure which might result in the stem opening at the base and shifting its base pairing after SSB binding nearby). As a result, approximately 30 nt SSB-free regions (i.e. the linkers structure in the functional SSB/ssDNA complex that was proposed by Grif®th et al. (1984) from electron microscopy observation and nuclease digestion analysis and was later con®rmed by Bujalowski & Lohman (1987) from a thermodynamic study) are generated on both sides of the stem-loop structure, between the stem-loop bound SSB tetramers and the 30 and 50 adjacent tetramers. The 50 CTG 30 pRNA initiation site is therefore ®xed in the 30 SSB-free region and another primase recognition site is arranged in the 50 SSB-free region. (2) Two primase molecules bind on each G4oric at speci®c DNA sequences (Stayton & Kornberg, 1983; and this work). One primase molecule (primase I) binds on the 50 CTG 30 containing sequence on the 30 near ¯ank and the other primase molecule (primase II) binds to another recognition DNA sequence on the 50 immediate ¯ank of the stem-loop structure. (3) A third SSB tetramer (SSB tetramer C) binding on the 30 distant ¯anking region, upstream of the pRNA initiation site in G4oric, is required for pRNA synthesis. Binding of a fourth SSB tetramer (SSB tetramer D) on the 50 far ¯anking region, although not essential for pRNA synthesis,

Table 1. Stoichiometry of binding of [3H]primase to [32P]G4oric/SSB complexes that were annealed with 50 or/and 30 antisense oligonucleotides In binding reactions Components and molar ratios (A) G4oric ‡ SSB ‡ primase (1:9:56) (B) G4oric ‡ 50 oligo ‡ SSB ‡ primase (1:14:21:47) 0 (C) G4oric ‡ {530 oligoes ‡ SSB ‡ primase (1:14(2):28:47) (D) G4oric ‡ 30 oligo ‡ SSB ‡ primase (1:14:21:47)

G4oric(fmol) 32 P Cold 740 880 880 880

4 4 2 2

[3H]primase (pmol) 42 42 42 42

cpma [3H]primase [32P]G4oric 100 57 0 55

733 424 161 206

In 3H/32P peak fractions Concentration (fmol) primase G4oric 65 34 0 32

35 35 22 30

Molar ratio primase/G4oric 1.9 1.0 0 1.1

a The cpm with cpm of background and 32P or 3H channel overlap subtracted. The background cpm were 20 for 3H and 30 for 32P. The channel overlaps in these experiments were approximately 5% of 32P cpm in the 3H channel and about 24% of 3H cpm in the 32P channel. [3H]primase and [32P]302 nt G4oric (plus cold 278 nt G4oric) were used to measure the binding number of primase molecules per G4oric ssDNA in the 32P and 3H peak fractions through gel ®ltration. The amount of [32P]G4oric ssDNA used in experiment C and D was half of that in experiments A and B. The experimental dates were day 1 for experiment A, day 10 for experiments B and C, and day 11 for experiment D.

Structure of Primase/SSB/G4oric Priming Complex

699

Figure 6. Diagram of the structure of the primase/SSB/G4oric priming complex. SSB A to SSB D represent SSB tetramers A to D.

increases activity of the G4oric/SSB complex to the wild-type level. It is clear that SSB plays an important role in forming the speci®c structure of the primase/SSB/ G4oric pRNA synthesis complex. The critical event appears to be binding of two SSB tetramers on the secondary structure of G4oric ssDNA, which creates SSB-free DNA regions on both the 30 and 50 sides of the structure. The pRNA initiation site and another primase recognition DNA sequence fall perfectly in the two SSB-free regions, which allow interaction with two primase molecules. Binding of SSB tetramers on the far 30 or 50 regions of hairpin increase pRNA synthesis mainly or partially, respectively. The function of SSB in the primase/ SSB/G4oric priming system, therefore, is to ®x the location of binding of primase to the speci®c DNA sequences on G4oric and to stabilize the interaction of primase with the ssDNA template. Binding of SSB to a stem-loop structure to ®x the recognition sequences for primase as we describe here may not be unique to the G4oric, but might be a general mechanism of making recognition DNA sequences available for primase or initiator proteins in other single-stranded phages. This hypothesis is based on the facts that SSB is absolutely required and initiation takes place close to or on the conserved hairpins in different single-stranded phage priming systems. In M13 and fd, the pRNA synthesis (by RNA polymerase instead of primase) requires SSB and starts from a hairpin structure (Geider & Kornberg, 1974; Geider et al., 1987). In the PriA-dependent primosomes such as fX174 in which the initiation starts by the binding of PriA protein on the primosome assembly site (pas) on the template, SSB is also required and pas regions always contain stem-loop structures (Greebaum & Marians, 1985; Masai et al., 1990b). In the DnaAdependant primosomes, it seems that such a close relationship between SSB binding and hairpin

structure is uncertain. However, an intensive study on plasmid R6K orig cloned in a single-stranded vector has shown that the proper binding of DnaA on the recognition sequence (DnaA box) in the origin of DNA replication was linked with SSB binding and these interactions were dependent on the existence of a hairpin structure (Masai et al., 1990a). The sequence ¯anking the 50 side of the small phage oric stem-loop structure, contrary to the sequence ¯anking the 30 side that contains the pRNA initiation site, has not been studied for primase interaction, except for some early footprinting data (Sims & Benz, 1980). Fourteen serial 50 G4oric deletion mutants described here, clearly demonstrated that 20 to 21 nucleotides 50 of stemloop III, especially the three adenine residues directly adjacent to the stem, are required for primase to synthesize pRNA normally. Blocking this sequence on the G4oric ssDNA template with complementary oligonucleotides con®rmed the requirement of this sequence for pRNA synthesis; the stoichiometric analysis further demonstrated that one primase molecule bound there. Two primase molecules bind to G4oric on two separated DNA sequences. The G4oric 50 binding site, however, does not contain the 50 CTG 30 sequence that is used to initiate pRNA synthesis on the 30 side. This suggests that the primase binding sequence may be more complex than a simple trinucleotide 50 CTG 30 sequence. On the 50 side, the cluster of adenine residues located on the 50 foot of stem-loop III may be involved in primase binding. These adenine residues are conserved in orics of all the single-stranded phages G4, St-1, fK and a3 and strongly protected from nuclease cleavage by primase binding in footprinting studies (Sims & Benz, 1980; Sun & Godson, 1993). A similar cluster of adenine residues are also present in the 30 ¯anking region immediately upstream of the

700 50 CTG 30 sequence (i.e. 50 CTGCAAAGCC 30 ) and these adenine residues were part of the minimum sequence of oligonucleotide templates active in pRNA synthesis (Swart & Griep, 1993). So far, there is no evidence that primase exists in solution as a dimer, and our gel ®ltration experiments shows that single molecules of primase can stably bind to the SSB/G4oric structure. The binding of two primase molecules to G4oric for normal pRNA synthesis might be explained by the structure of primase. Primase is a zinc metalloprotein (Stamford et al., 1992) and contains a single Nterminal zinc ®nger motif (Ilyina et al., 1992). DNA binding proteins (such as transcription factors) have at least two zinc ®ngers which ®t into the major groove of the double helix (Klug & Schwabe, 1995). A single zinc ®nger may not be suf®cient for primase to interact and correctly orient itself on G4oric DNA. In our experiments, we observed that primase can misread a sequence on a DNA template and incorporate a wrong ribo-50 triphosphate into pRNA chain. This was seen in the limited pRNA synthesis reactions which contained no substrate CTP and which were expected to give 8 nt pRNA with synthesis stopping at the ®rst guanine residue on G4oric template. Experimentally, however, pRNA chains were 9, 10 (minute) and 12 nt in length (data not shown), indicating that primase could read through the ®rst template guanine residue and stop at the next guanine residue, three bases downstream. When ddCTP was added to the reaction to enhance termination of pRNA synthesis, a single species of 9 nt pRNA was observed. Misincorporation of primase has been reported previously (Benz et al., 1980; Swart & Griep, 1993).

Materials and Methods Preparation of primase Primase was prepared from E.coli BL21 cells (Studier et al., 1990) containing the primase overproducing plasmid pGNG1 (Godson, 1991). After cell lysis, primase was precipitated with 40% to 50% NH4(SO4)2 and puri®ed by passage over a FPLC Mono Q 5/5 column (Pharmacia Biotech) (as described by Sun et al., 1994). [3H]primase was prepared from BL21 cells containing pGNG1 grown in 50 ml of M9 medium supplemented with all amino acids except alanine and valine. At A595 of 0.6 to 0.7, isopropyl-1-thio-b-D-galactopyranoside (0.4 mM ®nal concentration) was added to induce expression of primase. After 30 minutes, rifampicin (to 200 mg/ml) was added; 15 minutes later, 10 mCi each of [3H]alanine and [3H]valine (47 Ci/mmol, from Amersham Corp.) were added and three hours later the cells were harvested. [3H]primase was puri®ed as described above. The [3H]primase was quantitated by Bio-Rad protein assay and then checked by comparison of the Coomassie blue staining of primase with commercial standard proteins after gel electrophoresis. Both estimates were normally in agreement.

Structure of Primase/SSB/G4oric Priming Complex Preparation of viral DNA Viral ssDNA of M13/G4oric M (M13mp19 vector containing a 278 nt sequence of the negative strand of G4oric viral DNA; Sun & Godson, 1993) and f1R199/G4oric (f1R199 containing the plus strand of 278 nt G4oric DNA sequence; Sakai & Godson, 1985) were prepared as described in the references. The M13/G4oric (vector containing 278 nt sequence of the positive strand of G4oric viral DNA) RF DNA was prepared by using a routine method. Preparation of G4oric ssDNA and dsDNA fragments G4oric ssDNA fragments were generated by the methods we had described before (Sun & Godson, 1993). Wild-type G4oric 278 ssDNA was prepared by annealing of oligonucleotides that were complementary to the EcoRI cleavage sites of f1R199/G4oric and cleaving with EcoRI and then puri®ed following the above reference. Preparation of G4oric 302 and G4oric 149 ssDNA were described in that report. Different sized G4oric ssDNA fragments used in this work were prepared by primed synthesis using appropriate oligonucleotide primers on M13/G4oric M ssDNA template with the 30 terminus determined either by restriction enzyme cleavage or primer arrest. [32P]G4oric 302 nt ssDNA used for the gel ®ltration experiments was uniformly labeled with [a-32P]ATP during primed synthesis. The concentration of 32Plabeled G4oric ssDNA fragment was calculated from the [a-32P]AMP incorporated into synthesized G4oric ssDNA. This concentration was used as a standard to measure the amount of the cold G4oric 278 nt ssDNA by comparing their ethidium bromide ¯uorescence in a urea-polyacrylamide gel. Other [32P]G4oric ssDNA fragments were 50 end-labeled with [g-32P]ATP following a routine protocol. The dsDNA fragments of G4oric were generated from M13/G4oric RF DNA by PCR using Taq DNA polymerase (Boehringer Mannheim) and appropriate oligonucleotide primers. The standard temperature cycling conditions (1.5 minutes at 94 C, two minutes at 55 C and three minutes at 72 C) were used for ampli®cation of most G4oric DNA. G4oric fragments whose 50 or 30 end was located in a stem-loop structure were denatured at 96 C in PCR. Oligonucleotides The complementary oligonucleotides were chemically synthesized (DNAgency) and puri®ed by Centricon-10 concentrator (Amicon) before use. pRNA synthesis on ssDNA fragments The procedure was described by Hiasa et al. (1990). pRNA synthesis on heat denatured dsDNA G4oric templates: a new method A method of pRNA synthesis utilizing dsDNA fragments of G4oric instead of ssDNA as pRNA synthesis templates has been developed. This method worked well on small G4oric templates with 200 to 300 bp in length but did not work on as large as 7000 bp viral DNA. G4oric dsDNA fragments were generated by PCR and puri®ed using Gene Clean procedure (Bio 101 Inc.) to

701

Structure of Primase/SSB/G4oric Priming Complex remove PCR template DNA, excess primers and 30 deoxyribo 50 triphosphates. A G4oric dsDNA fragment (0.12 pmol) was mixed with 3 pmol SSB protein (purchased from United States Biochemical Co.) in pRNA synthesis buffer (20 mM TrisHCl (pH 7.5), 8 mM dithiothreitol, 8 mM MgCl2 and 4% (v/v) sucrose) in a 50 ml volume and the mixture was heated at 90 to 95 C for three minutes then fast cooled on ice. Primase (10 pmol), 100 mM ATP, 20 mM each CTP, GTP, and UTP, 20 mCi (6.6 pmol) of [a-32P]GTP (3000 Ci/mmol, DuPont, New England Nuclear) and 4 mg BSA were added to the reaction and the mixture was incubated at 30 C for 15 minutes. Synthesis was stopped by adding of 20 mM EDTA and the products were precipitated with ethanol. The pRNA was resuspended in 20 ml of 95% (v/v) formamide/dye and analyzed on 18% polyacrylamide (acrylamide: N0 , N0 methylene-bis-acrylamide, 19:1)/7 M urea gels. To identify the pRNA start site on G4oric, only three rNTPs (ATP, GTP and UTP) were added to the reaction, together with 20 mM ddCTP. The incubation time was extended to 30 minutes. The ef®ciency of pRNA synthesis from G4oric templates (i.e. molecules of pRNA synthesized per template DNA) was quantitated from [a-32P]GMP incorporation into pRNA chains that were excised from wet gels and counted in a Beckman liquid scintillation spectrometer. Using the known speci®c activity of added GTP (i.e. radioactive plus non-radioactive GTP) and the number of GMP incorporated into the synthesis products (i.e. 4 GMP for 9 nt pRNA synthesized in the presence of ddC and 10 GMP for full length pRNA), the number of pRNA molecules synthesized per DNA template can be calculated. For some experiments, the synthesis ef®ciency was measured by scanning the densities of pRNA bands from X-ray ®lm exposed to dried gels, using an LKB UltroScan XL ®lm scanner.

DNA molecule. The amount of DNase I required to cleave naked ssDNA was four times less than that used to cleave protein bound ssDNA. The 32P-labeled DNase I digests were puri®ed by phenol extraction and ethanol precipitation, and analyzed on 5% or 8% polyacrylamide/7 M urea sequencing gels (20 cm  40 cm).

Gel retardation experiment

Acknowledgments

32 P-labeled G4oric ssDNA (few ng) was mixed with SSB (approximately 0.1 to 1.0 pmol, depending on the amount required to saturate the DNA template) and primase (3.8 pmol in most experiments) in pRNA synthesis reaction buffer (see above) in 12.5 ml volume. The binding reactions were carried out at 30 C for ten minutes and the products immediately layered on a pre-run, 4% polyacrylamide gel (8 cm  8 cm; ratio of acrylamide to N0 , N0 -methylene-bis-acrylamide was 38:1). 8 mM MgCl2 was added to both the gel and the electrophoresis buffer (50 mM Tris-HCl (pH 8.0), 0.38 M glycine, 1 mM EDTA and 8 mM MgCl2) in order to maintain the same MgCl2 concentration as that in the binding reaction (Sun & Godson, 1996). Gels were run at 100 V.

We thank Dr Robert Schneider for valuable discussion about oligonucleotide blocking of SSB binding, Dr Nicholas Cowan for discussion of the binding reaction and gel ®ltration experiments and Dr Warren Jelinek for discussion regarding possibility of primase dimerization. We are grateful to Bernadette Yeaton of Bio-Rad Laboratories for technical advice on gel ®ltration. We especially thank Dr James Borowies for critical and careful reading of the manuscript. This work was supported by NIH grant GM32898 to G. N. G.

DNase I cleavage of protein/G4oric ssDNA binding complexes A 25 ml binding reaction contained approximately 10 ng of (50 -32P) end-labeled G4oric ssDNA, suf®cient SSB (1.3 to 2.1 pmol) to saturate the ssDNA, primase (12.5 pmol), and four rNTPs in pRNA synthesis buffer. The reaction was incubated at 30 C for ten minutes. The binding complexes were then digested with one to three units DNase I (Boehringer Mannheim) for 30 seconds at 30 C, which gave an average of single cleavage per

Gel filtration assay of primase binding The gel ®ltration followed Stayton & Kornberg (1983), except that Bio-Gel A-0.5 m agarose (®ne grade) was used because relatively low molecular masses of the SSB/small G4oric ssDNA complexes (approximately 5  105 Da) were used in this study. The amounts of ssDNA, SSB and primase were carefully measured using several different methods. As the speci®c radioactivity of 32 P-labeled G4oric 302 nt ssDNA (3  106 cpm/pmol) was much higher than that of 3H-labeled primase (1.7  103 cpm/pmol), the [32P]G4oric 302 nt ssDNA (<1 mg per ml ) was mixed with excess of cold G4oric 278 nt fragment (81 mg per ml) to lower the speci®c activity. To anneal complementary oligonucleotides to G4oric ssDNA, G4oric and the oligonucleotide(s) were mixed in 10 ml of 20 mM Tris-HCl (pH 7.5) and the mixture was heated to 85 C for two minutes then slowly cooled to 35 C. The partial ds G4oric DNA was used immediately for the binding reaction. 25 ml reaction containing G4oric ssDNA, SSB and primase (without adding rNTPs) in pRNA synthesis buffer was incubated at 30 C for 30 minutes. The sample was then applied to 5 ml Bio-Gel A-0.5 m column (0.75 cm  12 cm, equilibrated in 50 mM Tris-HCl (pH 7.5), containing 5 mM DTT, 10% (v/v) glycerol, 100 mg per ml BSA and 8 mM MgCl2) and was eluted with the same buffer in 90 ml fractions at 4 C. The 3H and 32P cpm were counted by liquid scintillation system (LS 7500, Beckman).

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Edited by M. Gottesman (Received 23 June 1997; received in revised form 2 September 1997; accepted 26 September 1997)