Mechanism of polymerase collision release from sliding clamps on the lagging strand
2009; Springer Nature; Volume: 28; Issue: 19 Linguagem: Inglês
10.1038/emboj.2009.233
ISSN1460-2075
AutoresRoxana E. Georgescu, Isabel Kurth, Nina Y. Yao, Jelena Stewart, Olga Yurieva, Mike O’Donnell,
Tópico(s)RNA Interference and Gene Delivery
ResumoArticle20 August 2009free access Mechanism of polymerase collision release from sliding clamps on the lagging strand Roxana E Georgescu Roxana E Georgescu Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Isabel Kurth Isabel Kurth Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Nina Y Yao Nina Y Yao Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Jelena Stewart Jelena Stewart Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Olga Yurieva Olga Yurieva Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Mike O'Donnell Corresponding Author Mike O'Donnell Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Roxana E Georgescu Roxana E Georgescu Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Isabel Kurth Isabel Kurth Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Nina Y Yao Nina Y Yao Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Jelena Stewart Jelena Stewart Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Olga Yurieva Olga Yurieva Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Mike O'Donnell Corresponding Author Mike O'Donnell Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA Search for more papers by this author Author Information Roxana E Georgescu1,‡, Isabel Kurth1,‡, Nina Y Yao1, Jelena Stewart1, Olga Yurieva1 and Mike O'Donnell 1 1Howard Hughes Medical Institute, The Rockefeller University, New York, NY, USA ‡These authors contributed equally to this work *Corresponding author. DNA Replication, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, Box 228, New York, NY 10021, USA. Tel.: +1 212 327 7255; Fax: +1 212 327 7253; E-mail: [email protected] The EMBO Journal (2009)28:2981-2991https://doi.org/10.1038/emboj.2009.233 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Replicative polymerases are tethered to DNA by sliding clamps for processive DNA synthesis. Despite attachment to a sliding clamp, the polymerase on the lagging strand must cycle on and off DNA for each Okazaki fragment. In the 'collision release' model, the lagging strand polymerase collides with the 5′ terminus of an earlier completed fragment, which triggers it to release from DNA and from the clamp. This report examines the mechanism of collision release by the Escherichia coli Pol III polymerase. We find that collision with a 5′ terminus does not trigger polymerase release. Instead, the loss of ssDNA on filling in a fragment triggers polymerase to release from the clamp and DNA. Two ssDNA-binding elements are involved, the τ subunit of the clamp loader complex and an OB domain within the DNA polymerase itself. The τ subunit acts as a switch to enhance polymerase binding at a primed site but not at a nick. The OB domain acts as a sensor that regulates the affinity of Pol III to the clamp in the presence of ssDNA. Introduction Chromosomal replicases are distinguished from repair DNA polymerases by their multi-subunit composition, rapid synthetic rate and high processivity (Benkovic et al, 2001; McHenry, 2003; O'Donnell, 2006). High processivity derives from a ring-shaped sliding clamp that is assembled onto DNA by a clamp loader machine. A DNA polymerase held to DNA by a mobile sliding clamp is nicely suited to highly processive synthesis on the leading strand of the replication fork but stands in contrast to the actions required on the lagging strand, which is synthesized as a series of Okazaki fragments in the direction opposite fork progression. Therefore, the lagging strand polymerase must rapidly dissociate after extension of each Okazaki fragment and recycle to a new upstream RNA primer to start the next fragment (Kornberg and Baker, 1992). Biochemical studies in diverse systems show that highly processive replicases rapidly recycle from one Okazaki fragment to the next, including Escherichia coli Pol III, eukaryotic Pol δ and bacteriophages T4 and T7 (O'Donnell, 1987; Hacker and Alberts, 1994; Stukenberg et al, 1994; Lee et al, 2006; Yang et al, 2006; Langston and O'Donnell, 2008). However, the mechanism that triggers the replicase to dissociate on reaching the end of an Okazaki fragment is poorly understood. In this report, we examine the detailed mechanism of collision release in the E. coli system. The E. coli replicase, DNA polymerase III holoenzyme (Pol III HE), consists of two molecules of the heterotrimeric Pol III core (α, DNA polymerase; ε, 3′–5′ exonuclease; and θ) that attach to the two τ subunits within the clamp loader (γ1τ2δ1δ′1χ1 ψ1) to form Pol III*; association of Pol III* with the β clamp forms the HE (McHenry, 2003; Johnson and O'Donnell, 2005). Studies in the E. coli system show that Pol III HE undergoes two types of recycling. In one polymerase-recycling process, Pol III HE remains tightly bound to DNA by the β clamp during processive synthesis, but Pol III* quickly releases from β on completing a DNA fragment, thereby releasing Pol III* to recycle to a new primed site (O'Donnell, 1987; Stukenberg et al, 1994). This recycling process is referred to as 'collision release' as Pol III HE collides with the 5′ terminus of a downstream Okazaki fragment. Once released, Pol III* rapidly reassociates with a new β clamp that has been assembled onto an RNA primer synthesized by primase. The second mechanism of polymerase recycling involves the release of Pol III* from β prematurely, before the Okazaki fragment is complete (Li and Marians, 2000; McInerney and O'Donnell, 2004). This mechanism is also observed in the T4 and T7 systems, in which it appears to be signalled by priming or clamp assembly on new RNA primers and is therefore referred to as 'premature release' or 'signalling release' (Li and Marians, 2000; Lee et al, 2006; Yang et al, 2006; Hamdan et al, 2009). The existence of two polymerase-recycling mechanisms on the lagging strand may reflect the importance of keeping lagging strand synthesis coupled with leading strand synthesis during chromosome duplication. Mechanistic studies of collision release in the E. coli system have shown that the τ subunit helps disengage Pol III from DNA and β on completing replication to a nick (Leu et al, 2003; Lopez de Saro et al, 2003). Although the exact mechanism by which τ functions is not known, it is suggested that τ may be located near the active site in the Pol III α subunit in which it can recognize DNA structural changes on completing an Okazaki fragment (Leu et al, 2003). In fact, the τ subunit binds the C-terminal region of α (Kim and McHenry, 1996), and structural studies show that this region is close to the polymerase active site (Bailey et al, 2006; Wing et al, 2008). The C-terminal region of E. coli Pol III α subunit contains two β-binding sites; one at the C-terminus and another 240 residues internal to the C-terminus (Lopez de Saro et al, 2003; Dohrmann and McHenry, 2005). The internal site is essential for processive function with β and is nicely positioned to bind the clamp in the α structure (Bailey et al, 2006; Wing et al, 2008). The extreme C-terminal β-binding site in α helps it to function with β and is required for function with τ (Dohrmann and McHenry, 2005; Lamers et al, 2006). This study focuses on the mechanism of collision release in the E. coli system. We first examine whether Pol III* recognizes the 5′ terminus to trigger collision release. The results show that 5′ terminal recognition is not involved in collision release. Hence, we shifted our focus to the role of ssDNA as a possible trigger for the release process, as ssDNA is lost on complete conversion to dsDNA. There exist two ssDNA-binding elements within Pol III*. One is an OB domain within α that binds template ssDNA and is positioned near the active site (Bailey et al, 2006; Wing et al, 2008). It is suggested that on completing an Okazaki fragment, the OB domain triggers a conformational change in Pol III that disconnects it from β (Wing et al, 2008). The other ssDNA-binding element in Pol III* is the τ subunit. Our earlier studies on τ show that it binds to ssDNA and has a function in collision release (Leu et al, 2003). Collision release requires Pol III to disconnect from two substrates, DNA and the β clamp. This study shows that the OB domain and τ subunit have distinct functions in collision release. The OB domain is a sensor that modulates the affinity of Pol III to the β clamp in response to ssDNA. Furthermore, we show that the τ subunit is a DNA switch that strengthens Pol III* binding to primed DNA, but not completed DNA (i.e. dsDNA or DNA with a nick). Thus, on completing an Okazaki fragment, both the τ switch and OB sensor are activated, and this relaxes the grip of Pol III* to DNA (τ subunit) and to the β clamp (OB domain), resulting in the release of Pol III* from β and DNA. Results Pol III HE does not recognize the 5′ terminus as a signal to disengage from β To address whether Pol III HE recognizes a 5′ terminal duplex as a signal to release from the β clamp and DNA, we blocked the 5′ terminus using a site-specific DNA-binding protein, Epstein–Barr virus origin binding protein 1 (EBNA1), which fully occludes its recognition sequence (Bochkarev et al, 1996). We placed the EBNA1 site at the extreme 5′ terminus of a DNA primer annealed to a 7.2-kb primed circular M13mp18 ssDNA (illustrated in Figure 1A). Pol III HE extends the primer full circle, resulting in a nick on reaching the 5′ terminus of the same primer. If Pol III HE must recognize the 5′ terminus to release from β, EBNA1 should prevent the recognition process and stop Pol III* from dissociating from β and DNA. Figure 1.Pol III HE does not recognize a 5′ terminus for collision release. (A) Pol III* and β were assembled onto a 7.2-kb primed M13mp18 ssDNA in the presence (or absence) of EBNA1, which binds to the extreme 5′ nucleotides of the 46-mer primed site. In a separate reaction, β is assembled onto a five-fold excess of a challenge primed M13Gori ssDNA (8.6 kb). Reactions were mixed, replication was initiated and timed aliquots were analysed in a native agarose gel. In control reactions, lanes 1–4, β was not assembled onto acceptor M13Gori DNA. Lanes 5–8 are reactions in the absence of EBNA1, and lanes 9–12 contained EBNA1. The 7.2- and 8.6-kb products were quantified using a phosphorimager and are shown in the plot as the ratio of replicated 8.6 kb acceptor RFII DNA over replicated 7.2 kb RFII donor DNA versus time. (B) The 5′ terminus of the primer is modified with either biotin (plus or minus streptavidin), or psoralen (see scheme). Reactions were performed and the 7.2- and 8.6-kb RFII products were quantified as described in (A). Download figure Download PowerPoint To monitor Pol III* release from β and DNA on completing replication, we performed a challenge assay using circular primed DNAs of different sizes. The 7.2-kb primed circular donor ssDNA contains an EBNA1 site. The challenge DNA is an 8.6-kb primed M13Gori ssDNA circle (O'Donnell, 1987; Stukenberg et al, 1994; Turner and O'Donnell, 1995). Pol III HE is first assembled onto the donor DNA in the presence or absence of EBNA1 along with only two dNTPs to prevent chain elongation. In a separate reaction, the β clamp is assembled onto a five-fold excess of 8.6 kb primed challenge DNA using the γ-complex clamp loader. The two reactions are then mixed and replication is initiated by adding the remaining dNTPs and α32P-dTTP. Pol III HE is processive and rapidly converts the 7.2-kb DNA into a circular duplex (i.e. RFII). If Pol III HE must recognize the 5′ terminus for collision release, then EBNA1 should prevent this recognition and Pol III* will not release and transfer to the 8.6-kb challenge DNA. On the other hand, if Pol III HE does not need to recognize the 5′ terminus it should still undergo collision release and cycle to the challenge DNA to produce an 8.6-kb RFII product. A control reaction confirms that the challenge DNA is only replicated when it is pre-loaded with a β clamp (Figure 1A, lanes 1–4). In the absence of EBNA1, Pol III* dissociates on completing the 7.2-kb DNA and readily cycles to the 8.6-kb challenge DNA (Figure 1A, lanes 5–8). In the presence of EBNA1, the rate of 8.6 kb RFII product formation is the same as in the absence of EBNA1 (Figure 1A, lanes 9–12 and quantitation to the right), indicating that occluding the 5′ terminus does not prevent the release of Pol III*. To exclude the possibility that Pol III HE binds the 5′ terminus by displacing EBNA1, we used 32P-labelled EBNA1 and analysed whether Pol III HE displaces it on replicating the circular DNA (Supplementary Figure S1). The results show that Pol III HE does not displace 32P-EBNA1 from DNA on completing replication, confirming that Pol III HE does not have access to the 5′ terminus in the challenge experiments. It is possible that Pol III* dissociates from β through a conformational change that occurs as a consequence of bumping into a protein block, rather than by triggering the collision release mechanism. Two lines of evidence indicate that a block to forward progression does not destabilize Pol III HE. First, Pol III* remains stably attached to β on DNA after encountering a template lesion, indicating that a sudden block to forward motion does not induce the polymerase to dissociate (McInerney and O'Donnell, 2007). Second, when an E. coli Pol III HE collides with an in-line RNA polymerase, the replisome remains bound to DNA, whereas it displaces the RNA polymerase and takes over the mRNA primer to continue DNA synthesis (Pomerantz and O'Donnell, 2008). This result indicates that encounter of a protein block per se does not induce the polymerase to release from DNA. Nevertheless, we cannot rigorously exclude that a conformational change occurs when the polymerase hits the EBNA1 block, inducing polymerase dissociation. To further support that 5′ end recognition is not required for collision release, we repeated the cycling experiments but used an oligonucleotide that was biotinylated at the 5′ terminus in the presence or absence of streptavidin (see scheme in Figure 1B). The result shows that after Pol III HE collides with the streptavidin bound to the 5′ terminus, it cycles to the challenge 8.6 kb DNA at the same rate as when it collides with an unmodified primer (Figure 1B). We also used a primer containing a bulky 5′ psoralen moiety and again observe transfer of polymerase to the challenge DNA (Figure 1B). These results support the conclusion that collision release by Pol III HE does not require recognition of a 5′ terminus. Pol III* can dissociate from the 3′ terminus before it dissociates from the β clamp If recognition of the 5′ terminus does not trigger collision release, it is possible that Pol III* detaches from the 3′ terminus before it dissociates from the β clamp. If so, Pol III*-β may remain together and slide along duplex DNA, as suggested by an earlier study (O'Donnell and Kornberg, 1985). To test whether Pol III* dissociates from β and DNA on colliding with a 5′ terminus, we observed its behaviour on encountering a short duplex in its path, during primer extension of a 5.4-kb ϕX174 ssDNA (see scheme in Figure 2A). We developed a DNA trap to capture any Pol III* that dissociates, thereby preventing it from reassociating with β on the downstream primer #2. The trap consists of 11-fold excess 7.2 kb M13mp18 ssDNA primed with a 3′ dideoxyoligonucleotide onto which β was assembled using the γ-complex clamp loader. The 3′ dideoxy terminus prevents extension and subsequent polymerase release. We used Pol III* containing an exonuclease-deficient ε subunit to prevent removal of the 3′ dideoxynucleotide. As a test of the trap, Pol III* and β were added to a mixture of the singly primed ϕX174 ssDNA and the excess trap M13mp18 ssDNA. The results (Figure 2A) show that the trap substrate is highly effective at sequestering free Pol III* from solution. Figure 2.Pol III HE releases DNA before it releases from β. (A) The reaction scheme to test effectiveness of a trap for free Pol III* is illustrated at the top. The trap is a 7.2-kb circular 3′dideoxy-terminated primed ssDNA, preloaded with a β clamp. Analysis of products in a native agarose gel at different amounts of added trap is shown to the left and quantification of DNA synthesis is shown in the plot to the right. (B) The experiment to test whether Pol III* stays bound to β after colliding with the downstream primer #2 is illustrated at the top. Pol III HE was assembled onto primer #1, and then the trap DNA (containing a preloaded clamp) was added along with primer #2 (as indicated). Replication was initiated and quenched at the indicated times followed by analysis in an alkaline agarose gel. Download figure Download PowerPoint If Pol III* releases from β on colliding with the 5′ terminus of primer #2, the polymerase will be sequestered by the trap, leaving only the 2.3-kb product. In contrast, if Pol III* remains bound to β on colliding with the 5′ terminus, the polymerase may slide over the duplex and extend primer #2 to form the 3.4-kb product. The experiment to examine whether Pol III* dissociates from β immediately on encountering a downstream primer is shown in Figure 2B. First, sub-stoichiometric Pol III HE was assembled onto the 5.4-kb ϕX174 ssDNA primed only with primer #1 (in the presence of two dNTPs to prevent extension). Then a second oligonucleotide (primer #2) is rapidly annealed to the ϕX174 ssDNA downstream of primer #1. Short oligonucleotides are known to anneal rapidly (<1 min) to SSB-coated ssDNA (O'Donnell and Kornberg, 1985). When dNTPs are added, Pol III HE will extend primer #1 until it collides with the 5′ terminus of downstream primer #2. If Pol III* releases from β and DNA, it will be trapped by β on the challenge DNA and only the 2-kb extension product of primer #1 will be observed. On the other hand, if Pol III* releases from DNA but stays attached to β, it will slide with β over primer #2, reattach to DNA and extend primer #2 to form the 3.4-kb segment. The results of the experiment show that in the presence of primer #2, a 2-kb product appears, rapidly followed by a 3.4-kb product (Figure 2B). This result indicates that on colliding with primer #2, Pol III* can stay attached to β, release from the 3′ terminus of the 2-kb product, and slide over primer #2, whereas staying bound to β. Quantitation of the replication products, shown below the gel, indicates that 35% of the time Pol III* remains bound to β and diffuses over primer #2. The presence of a small amount of full-length 5.4 kb product in reactions containing primer #2 is a background due to a small proportion of DNA templates that primer #2 does not hybridize to during the short 1-min annealing time. A control experiment without the trap shows about 75% extension of primer #2 after 60 s (Supplementary Figure S2). Hence, a significant amount of Pol III* remains bound to β, whereas it slides over primer #2. It is unclear whether the portion of Pol III* that dissociates from β does so before, or during β diffusion over the downstream primer. In summary, the results indicate that Pol III* does not necessarily release precisely at the site of collision with a 5′ terminus and supports the conclusion that the 5′ terminus is not the primary signal for collision release. The OB domain of Pol III α subunit is needed for processive function with the β clamp The experiments so far indicate that another trigger besides 5′ duplex recognition underlies collision release of Pol III* from β and DNA. On completing an Okazaki fragment, the ssDNA template strand is completely converted into duplex DNA, so the loss of contacts between the polymerase and ssDNA could bring about collision release. An invariant property of C-family DNA polymerases is the presence of an OB domain (Figure 3A). The crystal structure of Pol III α subunit reveals that the OB domain lies near the active site in which it can interact with template ssDNA just ahead of the polymerase (Bailey et al, 2006; Wing et al, 2008). This is also observed in the structure of Pol C from a gram-positive bacterium (Evans et al, 2008). Recent biochemical data show that the OB domain of E. coli α subunit binds ssDNA, but its function during DNA replication is not understood (McCauley et al, 2008). On the basis of the structure of Thermus aquaticus α subunit, it has been hypothesized that the OB domain in α may act as a sensor for the presence of the ssDNA template and that the loss of the OB–ssDNA interaction may trigger release of Pol III from β when synthesis of a fragment is completed (Bailey et al, 2006). Figure 3.The OB-fold within Pol III α subunit binds ssDNA and is needed for processive synthesis. (A) Scheme of the domain structure of Pol III α subunit. The expanded C-terminal region shows the OB domain and sequences that bind β and τ. (B) Fluorescence anisotropy DNA-binding assay. The isolated wt OB domain (left), or the mutant OB domain (right), is titrated into a reaction containing 5′-fluorescent labelled DNA. (C) Analysis of wt (squares) and OB-mutant (circles) Pol III core (left plot) and Pol III* (right plot) in β-independent assays using gapped DNA. (D) Native agarose gel product analysis of β-dependent replication assays using primed M13mp18 ssDNA and either wt Pol III core (lanes 1–4) or OB-mutant Pol III core (lanes 5–8). (E) As in (D) except using wt Pol III* (lanes 1–6) or OB-mutant Pol III* (lanes 7–12). Download figure Download PowerPoint To examine the role of the OB domain in Pol III HE function, we expressed and purified isolated wt and mutant OB domains (residues 962–1083) of Pol III α. The mutant form contained substitutions of three amino acids that are proposed to participate in ssDNA binding (R1004S, K1009S and R1010S) (Lamers et al, 2006). To determine whether these substitutions inactivate ssDNA binding, we used a DNA oligonucleotide labelled at the 5′ terminus with a fluorophore and measured the anisotropy change with increasing amounts of Pol III α OB domain. The results show that the wt OB domain binds to the ssDNA oligonucleotide with an apparent Kd value of 7.8±1.6 μM (Figure 3B, left panel), whereas ssDNA binding by the mutant OB domain was undetectable (Figure 3B, right panel). We also determined that neither the wt nor the mutant OB domain interacts with duplex DNA (Supplementary Figure S3). Next, we mutated the same OB-domain residues in the full-length α subunit and used it to reconstitute OB-mutant Pol III core and OB-mutant Pol III*. The reconstituted mutant complexes are nearly as active as their wt counterparts on activated calf thymus DNA. These substrates do not require processive activity for nucleotide incorporation, allowing to monitor catalytic activity independent of the β clamp (Figure 3C). This result indicates that ssDNA binding by the OB domain is not essential for DNA synthesis. On its own, the isolated OB-mutant α subunit is about half as active as wt α (Supplementary Figure S4), indicating that the additional subunits in Pol III core and Pol III* may stabilize the active site architecture or compensate for the reduced affinity of the OB-mutant Pol III α subunit to the DNA substrate. To examine the effect of α subunit OB-domain mutations on processive function with β, we assembled Pol III core-β or OB-mutant Pol III core-β on primed 7.2 kb M13mp18 ssDNA with only two dNTPs (the γ complex was used to assemble β onto DNA), then replication was initiated on adding the remaining dNTPs along with α32P-dTTP. Wild-type Pol III core-β rapidly converts the 7.2-kb substrate to a RFII circular duplex (Figure 3D, lanes 1–4). In contrast, the OB-mutant Pol III core-β yields only short products, indicating that the OB-mutant Pol III core is not processive with β (lanes 5–8, quantitation is in Supplementary Figure 5A). Hence, ssDNA binding by the OB domain of α is required for processive function of Pol III core with β. In summary, these results show that the OB domain of Pol III α binds ssDNA (Figure 3B) and that ssDNA binding is not required for intrinsic DNA polymerase activity (Figure 3C) but is required for processive function with the β clamp (Figure 3D). We show later in this report that ssDNA binding by the OB domain is required for optimal binding of α to β specifically at a primer/terminus, and that this accounts for the deficiency of the OB-mutant Pol III in function with β. The τ subunit partially rescues the OB-domain mutant The defect in processivity of the OB-mutant Pol III core may be explained by an altered affinity of Pol III core for DNA or the β clamp (or both). The τ subunit of the clamp loader is known to increase the binding of Pol III core to the β clamp on DNA (Stukenberg and O'Donnell, 1995), and therefore one may predict that the τ subunit will partially compensate for ssDNA-binding mutations in the OB domain of Pol III core. To examine this possibility, we tested the α OB mutant reconstituted into Pol III* (which contains τ) in β-dependent assays using primed M13mp18 ssDNA. The results, in Figure 3E lanes 7–12, show that the OB-mutant Pol III*-β produces full-length RFII duplex products, although it is less efficient and generates many short products compared with wt Pol III*-β (Figure 3E, lanes 1–6; see Supplementary Figure 5B for quantitation). Hence, the τ subunit partially rescues the defective processivity of the OB-mutant Pol III core in β-dependent synthesis. OB-mutant Pol III* is more deficient on the lagging strand than the leading strand The results obtained thus far indicate that the OB–ssDNA interaction is needed for processive synthesis of Pol III core with β, and thus one may expect that the OB mutant will not function properly on either the leading or the lagging strand. However, there may be no ssDNA on the leading strand, as leading strand synthesis does not require SSB (Mok and Marians, 1987). In this case, leading strand synthesis should not be hindered by OB mutations that eliminate ssDNA binding. Furthermore, the leading strand polymerase connects to DnaB helicase through the τ subunit (Dallmann et al, 2000), and this polymerase-helicase connection may confer processivity to the leading strand polymerase as shown earlier in the T4 and T7 systems (Xi et al, 2005; Johnson et al, 2007). To explore this issue, replisomes were reconstituted with DnaB and either the wt or OB-mutant Pol III HE on a synthetic 100-mer rolling circle replication fork substrate. Each strand of the synthetic minicircle contains only three nucleotides so that leading and lagging strands can be specifically labelled in separate reactions using either [α32P] dTTP or [α32P] dATP, respectively. The replisome is assembled by first loading DnaB on the 5′ dT40 tail of the minicircle, and then Pol III HE is assembled onto the leading strand in the presence of dCTP and dGTP to prevent fork movement (see scheme in Figure 4A). After 4 min, replication is initiated on adding dATP, dTTP, SSB, DnaG primase, the four rNTPs and the appropriate radiolabelled dNTP. Timed aliquots are analysed in alkaline agarose gels (Figure 4B). Figure 4.The OB-mutant Pol III HE is deficient in lagging strand synthesis. (A) Scheme of replisome assembly on a minicircle replication fork substrate using DnaB helicase and wt or OB-mutant Pol III HE (B) Leading (left panel) and lagging (right panel) strands can be selectively labelled depending on whether α-32P dATP or α-32P dTTP is present during synthesis. Aliquots were removed at the indicated times and analysed in a 1.2% alkaline agarose gel followed by autoradiography. The relative level of DNA synthesis at 320 s is shown at the bottom of the gel. Download figure Download PowerPoint The results show that replisomes containing the OB-mutant Pol III HE give lower synthesis on both leading and lagging strands compared with wt Pol III HE (Figure 4B), yet the lagging strand is more affected by the OB mutation than the leading strand. The fact that leading strand synthesis is impaired by the OB mutation suggests that ssDNA may be present on the leading strand ahead of the polymerase. Alternatively, the OB mutations may impair assembly of Pol III HE into the replisome or alter a property of Pol III HE function in the context of the replisome that is not divulged in assays using primed M13mp18 ssDNA. Earlier studies have shown that Okazaki fragment size is determined by fork speed and primase concentration (Wu et al, 1992). The gel analysis in Figure 4B shows that the speed of the leading strand OB-mutant replisome is the same as the wt replisome. Therefore, as primase concentration is held constant in these experiments, the lagging strand should be primed at the same frequency and should produce similar sized Okazaki fragments. However, the results clearly show a decrease in Okazaki fragment size from 1 to 0.5 kb for the OB-mutant replisome (Figure 4B, right). Furthermore, quantitation of the products shows a
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