The phi X174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition
1997; Springer Nature; Volume: 16; Issue: 22 Linguagem: Inglês
10.1093/emboj/16.22.6886
ISSN1460-2075
AutoresJessica M. Jones, Hiroshi Nakai,
Tópico(s)CRISPR and Genetic Engineering
ResumoArticle15 November 1997free access The φX174-type primosome promotes replisome assembly at the site of recombination in bacteriophage Mu transposition Jessica M. Jones Jessica M. Jones Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007 USA Search for more papers by this author Hiroshi Nakai Corresponding Author Hiroshi Nakai Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007 USA Search for more papers by this author Jessica M. Jones Jessica M. Jones Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007 USA Search for more papers by this author Hiroshi Nakai Corresponding Author Hiroshi Nakai Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007 USA Search for more papers by this author Author Information Jessica M. Jones1 and Hiroshi Nakai 1 1Department of Biochemistry and Molecular Biology, Georgetown University Medical Center, 3900 Reservoir Rd NW, Washington, DC, 20007 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1997)16:6886-6895https://doi.org/10.1093/emboj/16.22.6886 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Initiation of Escherichia coli DNA synthesis primed by homologous recombination is believed to require the φX174-type primosome, a mobile priming apparatus assembled without the initiator protein DnaA. We show that this primosome plays an essential role in bacteriophage Mu DNA replication by transposition. Upon promoting transfer of Mu ends to target DNA, the Mu transpososome undergoes transition to a pre-replisome that permits initiation of DNA synthesis only in the presence of primosome assembly proteins PriA, DnaT, DnaB and DnaC. These assembly proteins promote the engagement of primase and DNA polymerase III holoenzyme, initiating semi-discontinuous replication preferentially at the Mu left end. The results indicate that these proteins play a crucial role in promoting replisome assembly on a recombination intermediate. Introduction Coupling of DNA synthesis to recombination is an important mechanism involved in DNA repair, genetic exchange and chromosomal replication. Growing evidence suggests interdependence between chromosomal replication and homologous recombination, DNA replication participating in the formation of recombinants and homologous recombination leading to initiation of chromosomal replication (Kogoma et al., 1996). Involvement of the primosome assembly protein PriA in both recombinant formation and recombination-dependent DNA replication in Escherichia coli has suggested that it may be part of an apparatus for linking strand exchange with DNA synthesis. PriA is a constituent of the φX174-type primosome, which originally was characterized for its function in converting single-stranded phage φX174 DNA to the duplex replicative form (Kornberg and Baker, 1992). It is distinguished from the oriC-type primosome by the involvement of host-encoded PriA, PriB, PriC and DnaT proteins in primosome assembly instead of the initiator protein DnaA, which promotes replisome assembly at the bacterial origin of replication. In φX174 replication, PriA binds to the unique primosome assembly site (PAS) on single-stranded phage DNA and recruits PriB, PriC and DnaT (Shlomai and Kornberg, 1980; Liu et al., 1996; Ng and Marians, 1996a). With the assistance of the associated matchmaker DnaC, DnaB helicase is then delivered to the complex to form the preprimosome. DnaB within this mobile apparatus interacts transiently with primase to form the primosome (Tougo et al., 1994; Ng and Marians, 1996b), which catalyzes synthesis of RNA primers at many sites on the template to initiate DNA synthesis by the DNA polymerase (pol) III holoenzyme (Ng and Marians, 1996b). PriA's ability to promote primosome assembly plays an important role in DnaA-independent DNA synthesis such as pBR322 replication (Minden and Marians, 1985). On a preformed replication fork, which is a circular duplex with a single-stranded tail, PriA can promote the assembly of a replisome that catalyzes leading and lagging strand synthesis if a PAS is present on the tail (Wu et al., 1992). However, the φX-type primosome is not necessarily required for replication of the bacterial chromosome. DNA replication initiated at oriC can be reconstituted in vitro without the PriA, PriB, PriC and DnaT proteins (Kaguni and Kornberg, 1984). Strains with priA null mutations are viable although they display characteristics of slow growth, filamentous structure, increased sensitivity to DNA-damaging agents and a constantly induced SOS system (Lee and Kornberg, 1991; Nurse et al., 1991). It has been suggested that the φX-type primosome may be required for reinitiation should the replisome stall (Nurse et al., 1991). Recent evidence demonstrates that priA null strains show poor assimilation of genetic markers by homologous recombination and are defective in DNA double strand break repair (Kogoma et al., 1996). They are also deficient in inducible and constitutive stable DNA replication (iSDR and cSDR) (Masai et al., 1994), forms of chromosomal replication which occur independently of the DnaA protein. Since iSDR is dependent on homologous recombination functions, a model has been proposed for the function of the φX-type primosome in coupling recombination with replication (Asai and Kogoma, 1994; Kogoma, 1996). The potential replication fork is produced when an invading strand displaces one strand of a duplex to form a D-loop structure (Eggleston and West, 1996) and provides the potential primer for leading strand synthesis. The φX-type primosome is assembled on the single-stranded region within the D-loop, promoting replisome assembly and establishing a replication fork (Kogoma, 1996). In support of this hypothesis, DnaT and DnaC, which are also involved in the assembly of the φX-type primosome, are required for iSDR as well (Masai and Arai, 1988). In addition, PriA can bind to D-loops and related DNA structures (McGlynn et al., 1997). However, the ability of the φX-type primosome to promote initiation of replication on a natural recombination intermediate has heretofore not been demonstrated. Phage Mu DNA replication by transposition resembles the hypothesized mechanisms for DNA replication coupled to homologous recombination. In Mu transposition, strand exchange is catalyzed by the phage-encoded transposase MuA (for reviews, see Mizuuchi, 1992; Chaconas et al., 1996; Lavoie and Chaconas, 1996). Monomeric MuA binds to specific sequences at each Mu end (Craigie et al., 1984; Kuo et al., 1991), assembling into a tetramer that holds together the two ends (Lavoie et al., 1991). This transpososome introduces a nick at each end, and the resulting 3′-hydroxyl groups are transferred to target DNA (Craigie and Mizuuchi, 1987; Surette et al., 1987; Mizuuchi et al., 1992), producing a branched DNA structure with a potential replication fork at each Mu end. A specific set of host proteins is required to replicate Mu DNA on this strand transfer intermediate, and MuA plays a key role in controlling access of host proteins to the two potential replication forks (Kruklitis and Nakai, 1994; Nakai and Kruklitis, 1995; Kruklitis et al., 1996). Oligomeric MuA remains tightly bound to both Mu ends in a nucleoprotein complex known as the strand transfer complex (STC1) or type II transpososome (Surette et al., 1987; Lavoie et al., 1991). A group of host factors called Mu replication factors α (MRFα), which includes the molecular chaperone ClpX and at least one additional component (MRFα2) (Kruklitis et al., 1996), removes MuA from STC1 to form a prereplisome, a nucleoprotein complex (STC3) that only allows initiation of Mu DNA synthesis by a specific set of host factors (Nakai and Kruklitis, 1995). These factors include replication proteins such as DnaB, DnaC and DNA pol III holoenzyme, which are known to be required for Mu DNA synthesis in vivo, and a group of host factors called MRFβ, previously used in the reconstituted system in partially purified form. In this study, we identify the host factors in MRFβ as PriA, PriB and DnaT. We characterize the function of these proteins in promoting Mu replication on the Mu strand transfer intermediate. Results Mu replication by transposition in vivo is dependent on the priA gene function We examined the ability of Mu to grow in E.coli strains with inactivating mutations in the priA gene. Two E.coli strains with priA null mutations (PriA−) supported Mu lysogenization but were unable to support lytic growth (Table I). The ability to support Mu lytic growth was restored by transformation with a plasmid expressing PriA (Table I). Table 1. PriA− Escherichia coli hosts can support Mu lysogenization but not lytic development Host straina Relevant trait Mu plating efficiency Frequency of lysogenization EL501 PriA+ 1 8 ×10−3 EL500 PriA− <10−7 0.7×10−3 EL502 PriA+ 0.8 not determined AT3327 PriA+ 1 4 ×10−3 AT3327 priA1::kan PriA− 1010 plaque-forming units (p.f.u.) per ml] whereas the PriA− lysate had no detectable titer (<103 p.f.u. per ml). Southern blot analysis of DNA isolated from the induced PriA+ Mu lysogen (Figure 1A, lanes 1–4) indicated that Mu DNA was amplified at least 25-fold relative to a host-specific marker (dnaA) before lysis (Figure 1B). No amplification was detected in the induced PriA− lysogen (Figure 1A, lanes 5–8, and Figure 1B) even though reconstruction experiments indicated that as little as a 2-fold increase in Mu DNA could be detected using this Southern blot technique (data not shown). These results indicate that Mu was unable to undergo even one round of replication by transposition in vivo in the absence of PriA. Figure 1.Requirement for the priA function in bacteriophage Mu DNA replication in vivo. (A) Southern blot of DNA prepared from induced cultures of Mu lysogens AT3978 (PriA+) and AT3978 priA1::kan (PriA−) probed with Mu-specific and E.coli dnaA-specific sequences. (B) Quantitation of Mu DNA amplification relative to an E. coli-specific marker (dnaA). Solid and open arrows indicate the time at which lysis occurred for the PriA+ and PriA− Mu lysogens, respectively. Download figure Download PowerPoint PriA and additional φX-type primosome constituents are required for Mu DNA replication in vitro In the in vitro transposition system, STC1 is formed using a supercoiled plasmid bearing a mini-Mu element as donor substrate and a second plasmid as target (Mizuuchi, 1983). Mu DNA in STC1 can be replicated to form a cointegrate using a reconstituted system composed of an eight-protein system [DnaB, DnaC, primase, DNA pol III holoenzyme, DNA pol I, DNA gyrase, single-strand binding protein (SSB) and DNA ligase] supplemented with MRFα (or ClpX and MRFα2) and MRFβ (Kruklitis and Nakai, 1994; Nakai and Kruklitis, 1995). MRFα and MRFβ can be supplied separately (each as fraction III) or together in a crude enzyme fraction (fraction II). We determined whether PriA was an essential component of this system. The eight-protein system supplemented with fraction II from a PriA− E.coli strain did not support Mu DNA replication (Figure 2A). The addition of purified PriA restored only low levels of replication activity, while the addition of both PriA and DnaT restored activity to that obtained with fraction II from a wild-type strain, suggesting that Mu DNA replication was dependent on both PriA and DnaT and that our PriA− fraction II was also deficient in DnaT activity. Using a reconstituted assay for the replication of φX174 single-stranded DNA, we found that our PriA− fraction II was indeed partially deficient in DnaT activity relative to a fraction II from a PriA+ strain (data not shown). Figure 2.Requirement for PriA and additional primosome proteins in the reconstituted Mu replication system. (A) Replication was catalyzed on STC1 (pXP10 target DNA) in the eight-protein system supplemented with the indicated proteins and with varying amounts of a crude enzyme fraction (fraction II) prepared from a PriA+ (WT) or PriA− E.coli strain. (B) Replication was catalyzed on STC1 in the eight-protein system supplemented with MRFα, purified PriA, as indicated, and varying amounts of MRFβ prepared from PriA+ (WT) or PriA− strains. (C) Replication was catalyzed on STC1 in the eight-protein system supplemented with MRFβ(WT) or purified PriA, PriB and DnaT, as indicated, and with varying amounts of MRFα(PriA−). Download figure Download PowerPoint PriA was a necessary component of MRFβ which provides complementing activity in the reconstituted Mu replication system. While an MRFα fraction III prepared from a PriA− strain had complementing activity comparable with MRFα from a PriA+ strain (data not shown), the MRFβ fraction III prepared from a PriA− strain showed only background levels of activity (Figure 2B). Unlike the PriA− fraction II, full activity was restored to MRFβ (PriA− fraction III) by the addition of purified PriA alone (Figure 2B). The specific activity of MRFβ is increased 10- to 15-fold during preparation of fraction III, and therefore the enrichment of low levels of DnaT in fraction II as well as removal of unwanted proteins most likely yielded a MRFβ(PriA−) fraction with sufficient DnaT activity to promote high levels of Mu DNA replication. MRFβ could be replaced by purified PriA, PriB and DnaT (Figure 2C). Cointegrate production was absolutely dependent on PriA, DnaBC and MRFα as well as the φX components PriB and DnaT (Table II). The small amounts of cointegrate production apparent when either PriB or DnaT was omitted individually are most likely due to low levels of PriB and DnaT in the MRFα fraction, detected using the reconstituted φX174 replication assay (data not shown). The lack of any replication when both are omitted (Table II) strongly supports the conclusion that PriA is not acting independently of PriB and DnaT during Mu DNA replication but is assembling a multi-component primosome like the one characterized in φX174 replication. We could not determine the dependence of Mu replication on PriC because high levels of PriC activity were present in the MRFα fraction (data not shown). MRFα cannot be replaced with purified PriC and ClpX (Table II), indicating that at least one additional factor besides these two proteins is an essential MRFα component. Table 2. Requirement for φX-type primosome components and MRFα in cointegrate formation Component omitteda pmolb Co (%)c None 185 100 MRFα 0 <1 MRFα (ClpX and PriC added) 0 <1 PriA 0 <1 DnaBC 0 <1 PriB and DnaT 0 95% of the strand transfer products were converted to cointegrates, was set arbitrarily at 100. The φX-type primosome supports initiation of semi-discontinuous DNA synthesis with initial preference for the Mu left end Replication of full-length (37 kb) Mu DNA in induced lysogens proceeds semi-discontinuously (Higgins et al., 1983), with DNA synthesis in vivo initiating 80–90% of the time at the left end of full-length Mu (Wijffelman and van de Putte, 1977; Goosen, 1978; Pato and Waggoner, 1987). However, initiation of mini-Mu replication in vivo takes place at the left end only ∼50% of the time (Harshey et al., 1982; Résibois et al., 1982a,b, 1984). We examined these properties in the reconstituted Mu replication system. To distinguish between leading and lagging strand synthesis and between initiation at the Mu left and right ends, STC1 was replicated in a six-protein system (the eight-protein system lacking DNA pol I and ligase) supplemented with MRFα, PriA, PriB, PriC and DnaT. Products were digested with a restriction enzyme that cleaves within the donor vector near the Mu left end (Figure 3A). Leading strands corresponding to initiation at the left or right ends as well as Okazaki fragments from lagging strand synthesis could be distinguished by size on a denaturing agarose gel. To ensure examination of leading and lagging strand synthesis associated with cointegrate formation, linearized cointegrate products were first purified from a native agarose gel prior to separation by denaturing gel electrophoresis. Figure 3.Replication of STC proceeds by semi-discontinuous DNA synthesis. (A) BamHI and NdeI cleave asymmetrically in the donor vector but not within the mini-Mu element or the transposition target. Cleavage of unligated replication products with one of these enzymes (e.g. BamHI) results in a unique series of labeled DNA fragments whose lengths depend on the mode of replication: initiation of leading and lagging strand synthesis from the left (i) or right ends (ii) or initiation of leading strand synthesis from the primers at both ends (iii). (B) and (C) Replication on STC1 (φX174 RFI target DNA) was conducted in the six-protein system (lacking DNA pol I and ligase) (B) or the eight-protein system (C) supplemented with MRFα(PriA−), PriA, PriB and DnaT. Full-length cointegrate products linearized with BamHI were purified by native gel electrophoresis and then resolved on a 0.6% alkaline agarose gel, which was dried for phosphorimagery. Linear scans of the radiolabeled products in each lane are shown. Peaks corresponding to unit length cointegrate (Co), leading strand products resulting from initiation at the Mu right (CoR) and left (CoL) ends and products of lagging strands synthesis were identified based on their migration relative to molecular weight standards. Download figure Download PowerPoint We confirmed the presence of short products (1–3 kb) consistent with lagging strand synthesis in the isolated co-integrate products (Figure 3B), with leading and lagging strand synthesis accounting for roughly equal amounts of nucleotide incorporation. The addition of DNA pol I and ligase shifted all products to the unit length of the co-integrate (Figure 3C), supporting the conclusion that the short products were indeed Okazaki fragments. Quantitation of the products of leading strand initiation from the left and right ends in these isolated cointegrates revealed only a small bias for initiation from the left end. The relative frequency of leading strand synthesis initiating at the left and right ends of mini-Mu was determined in vitro in this experiment from all replication products that had accumulated at the completion of the reaction (30 min) and in vivo in previous work (Résibois et al., 1984) from all products that had accumulated late in development. To determine whether earlier replication products in vitro reflect the left end bias seen with full-length Mu in vivo, we examined the kinetics of initiation at the left and right ends. Reactions were allowed to proceed for 5–30 min, and products were digested with restriction enzymes that cleave in the donor vector either very near the Mu left (BamHI) or right end (NdeI) to distinguish leading strands corresponding to initiation at the left or right ends on a denaturing agarose gel (see Figure 3A). Full-length products corresponding to leading strand synthesis across the entire mini-Mu element were first evident at 10 min. Quantitation of cointegrate products digested with BamHI or NdeI (Figure 4A) revealed that 90–100% of cointegrates formed at 10 min corresponded to initiation at the left end of Mu (Figure 4B), indicating that the initial rounds of replication do reflect a left end bias. Products of right end initiation accumulated more slowly, so that by 30 min they accounted for 25–45% of the products (Figure 4B). Thus, some feature of STC3 or the DNA template may permit the replisome to be assembled more readily at the left end. All of these results indicate that Mu DNA replication reconstituted with the φX174-type primosome reflects characteristics of Mu DNA replication observed in vivo. Figure 4.Replication on STC initiates preferentially from the left end of Mu. (A) Replication on STC1 (φX174 RFI target DNA) was allowed to proceed for 5–30 min in the six-protein system (lacking DNA pol I and ligase) supplemented with MRFα(PriA−), PriA, PriB and DnaT. Cointegrate products were linearized with BamHI or NdeI and resolved on a 0.6% alkaline agarose gel, which was dried for phosphorimagery. Linear scans of the radiolabeled leading strand products from the 10, 20 and 30 min reactions are shown. Peaks corresponding to leading strand products resulting from initiation at the Mu right (CoR) and left (CoL) ends were identified based on their migration relative to molecular weight standards. Total deoxynucleotide incorporation (pmol) in each reaction is indicated; scans have been normalized for total cointegrate formation. (B) The percentage of total leading strand synthesis initiating at the Mu left end was quantitated by phosphorimagery. Results are the average of three independent trials, including one in which products were digested with NdeI and two in which products were digested with BamHI; standard deviation of the mean is indicated by error bars. Download figure Download PowerPoint φX-type primosome constituents promote engagement of DNA pol III holoenzyme on the recombined substrate Mu DNA synthesis can initiate without MRFα, MRFβ, DnaB, DnaC and DNA pol III holoenzyme on the deproteinized strand transfer product (Kruklitis and Nakai, 1994; Nakai and Kruklitis, 1995), especially when DNA pol I (or the Klenow fragment) is present at high levels (Figure 5B, lane 1). We determined whether DNA pol III holoenzyme (prepared from a UvrD− strain so that it is not contaminated with helicase II) can catalyze Mu DNA synthesis on the deproteinized strand transfer product when PriA, PriB and DnaT are absent. The deproteinized template was incubated for 15–60 min in the six-protein system (in the absence of DNA pol I and ligase), and products were cleaved within the donor vector (Figure 3A) so that extension from the two ends could be distinguished. Even after 30 min, no DNA synthesis was catalyzed on the deproteinized template in the six-protein system alone (Figure 5A, lane 1). When the six-protein system was supplemented with high levels of the DNA pol I Klenow fragment, extension of the leading strand primers at both ends proceeded slowly, consistent with the low processivity and distributive action of pol I. These primers were extended only 0.2–0.4 kb by 15 min (Figure 5A, lane 2), gradually being extended 1 kb or more by 60 min (Figure 5A, lane 5). Few or no products corresponding to complete replication of the mini-Mu element were formed even after 60 min. Moreover, the same level of DNA synthesis was catalyzed if DnaB and pol III holoenzyme were not present together with pol I (Figure 5B, lane 1). These results indicate that DnaB and DNA pol III holoenzyme are not engaged on the deproteinized template under these conditions. Figure 5.φX-type primosome constituents promote engagement of DNA pol III holoenzyme on the deproteinized strand transfer product. (A) Replication was conducted on the deproteinized strand transfer product (φX174 RFI target DNA) in the six-protein system supplemented with the DNA pol I Klenow fragment (100 U/ml) for 15–60 min (lane 1: six-protein system alone, 30 min). Products were digested with BamHI and resolved on a 0.6% alkaline agarose gel. The length of the replication products increases with time as the leading strand primers are slowly extended by Klenow. Total deoxynucleotide incorporation (pmol) in each reaction is indicated. For reference, the positions of unextended leading strand primers from the strand transfer intermediate (S) and of fully extended leading strands from the co-integrate (Co) resulting from initiation at the Mu left (CoL, SL) and right (CoR, SR) ends are indicated; replication products in this reaction did not reach full length. (B) Replication was conducted on the deproteinized strand transfer product (f1 RFI target DNA) in the eight-protein system supplemented with PriA, PriB, PriC, DnaT and additional DNA pol I (2 U/ml). Proteins were omitted as indicated (lane 1: replication by 2 U/ml DNA pol I in the absence of DnaBC, PriABC, DnaT and DNA pol III). Products were digested with EcoRI and resolved on a 0.6% alkaline agarose gel. Positions of the full-length cointegrate (Co), unreplicated strand transfer intermediates (SL and SR) and leading strand primers extended by DNA pol I (Ex) are shown. (C) Total deoxynucleotide incorporation (pmol) was determined by counting one-tenth of each reaction mixture (white bars). The remaining products were resolved on a 0.6% alkaline agarose gel. The amount of cointegrates was quantitated by phosphorimagery (shaded bars). The level of cointegrates formed in the complete reaction (no components omitted), in which ∼60% of the strand transfer products were converted to cointegrates, was set arbitrarily at 100. Results are the average of two independent experiments. Download figure Download PowerPoint However, when PriA, PriB, PriC and DnaT were added to the reaction mixture that included DNA pol I, full-length cointegrates were formed in 30 min (Figure 5B, lane 2). DNA ligase was included in these reactions so that full-length cointegrates could be easily distinguishable from the shorter, 30 min extension products of DNA pol I (Figure 5B, cf. Co and Ex). Quantitation of cointegrate production revealed that under these conditions at least 90% of the cointegrate products were dependent on not only PriA and DnaT but also on the DnaBC complex and pol III holoenzyme (Figure 5C). In separate experiments, we determined that cointegrate production was dependent on both DnaB and DnaC when they were added individually (data not shown). Therefore, the PriA-dependent replication pathway engages DnaB helicase and pol III holoenzyme to replicate Mu DNA rapidly on the strand transfer product. Extension of the leading strand primer by DNA pol I is not essential for PriA-dependent DNA synthesis on the Mu strand transfer intermediate In pBR322 replication, an RNA polymerase transcript that primes DNA synthesis at the origin must be extended by DNA pol I to form a D-loop and expose a PAS on the displaced single strand to maximize PriA-promoted assembly of the pre-primosome (Minden and Marians, 1985). On the Mu strand transfer intermediate, there is no single-stranded region on the lagging strand side of each fork potentially to serve as a binding site for the pre-primosome (see Figure 7A). Although DNA pol I can extend the leading strand at each Mu end of the deproteinized template to expose single-stranded DNA, it was not essential for PriA-dependent cointegrate formation (Figure 5C). Its presence did increase the level of nucleotide incorporation and cointegrate formation by ∼2-fold, suggesting the possibility that the efficiency of preprimosome assembly can be maximized by limited extension of the leading strand primers. When DNA synthesis was catalyzed on STC1, the leading strand primers were not extended at all unless all required replication proteins including PriA, DnaT and MRFα were present (Figure 6, lane 1). When PriA or DnaBC was omitted, no cointegrates were formed, and the leading strand primers could not be extended by high levels of DNA pol I (Figure 6, lanes 2 and 3) as they were on the deproteinized template (lane 4). Whereas 400–500 nucleotides were incorporated per deproteinized template in 30 min, the amount of nucleotide incorporation during this time on the STC without PriA or DnaBC was below detectable levels, which correspond to <10 nucleotides being incorporated per template. This level of nucleotide incorporation by itself is unlikely to produce a duplex opening sufficient to promote primosome assembly. When the DNA duplex at a ColE1-type plasmid origin is opened by an R-loop, a single-stranded region with a minimum of 40 bases must be exposed to activate DNA synthesis in the absence of DNA pol I (Masukata et al., 1987). Together with previous findings that the polymerase activity of DNA pol I is not required to initiate DNA synthesis on STC (Kruklitis and Nakai, 1994), our results in
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