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Defining the position of the switches between replicative and bypass DNA polymerases

2004; Springer Nature; Volume: 23; Issue: 21 Linguagem: Inglês

10.1038/sj.emboj.7600438

1460-2075

Shingo Fujii, Robert P. Fuchs,

CRISPR and Genetic Engineering

Article7 October 2004free access Defining the position of the switches between replicative and bypass DNA polymerases Shingo Fujii Shingo Fujii UPR 9003 du CNRS, Cancerogenese et Mutagenese Moleculaire et Structurale, ESBS, Blvd S Brant Strasbourg, Illkirch, France Search for more papers by this author Robert P Fuchs Corresponding Author Robert P Fuchs Search for more papers by this author Shingo Fujii Shingo Fujii UPR 9003 du CNRS, Cancerogenese et Mutagenese Moleculaire et Structurale, ESBS, Blvd S Brant Strasbourg, Illkirch, France Search for more papers by this author Robert P Fuchs Corresponding Author Robert P Fuchs Search for more papers by this author Author Information Shingo Fujii1 and Robert P Fuchs 1UPR 9003 du CNRS, Cancerogenese et Mutagenese Moleculaire et Structurale, ESBS, Blvd S Brant Strasbourg, Illkirch, France *Corresponding author. UPR 9003 Cancerogenese & Mutagenese, Moleculaire et Structurale CNRS, ESBS Pole API, Boulevard Sebastien Brant, 67400 Illkirch-Graffenstaden, France. Tel.: +33 390 244 688; Fax: +33 390 244 686; E-mail: [email protected] The EMBO Journal (2004)23:4342-4352https://doi.org/10.1038/sj.emboj.7600438 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Cells contain specialized DNA polymerases that are able to copy past lesions with an associated risk of generating mutations, the major cause of cancer. Here, we reconstitute translesion synthesis (TLS) using the replicative (Pol III) and major bypass (Pol V) DNA polymerases from Escherichia coli in the presence of accessory factors. When the replicative polymerase disconnects from the template in the vicinity of a lesion, Pol V binds the blocked replication intermediate and forms a stable complex by means of a dual interaction with the tip of the RecA filament and the β-clamp, the processivity factor donated by the blocked Pol III holoenzyme. Both interactions are required to confer to Pol V the processivity that will allow it synthesize, in a single binding event, a TLS patch long enough to support further extension by Pol III. In the absence of these accessory factors, the patch synthesized by Pol V is too short, being degraded by the Pol III-associated exonuclease activity that senses the distortion induced by the lesion, thus leading to an aborted bypass process. Introduction In all living cells, DNA continuously incurs damages by endogenous and exogenous agents. Excision repair pathways remove lesions in an essentially error-free way (Friedberg et al, 1995). Despite these robust repair pathways, residual levels of lesions persist in DNA thus impairing the progression of replicative DNA polymerases. Escherichia coli cells respond to stress by inducing coregulated sets of genes, such as the SOS regulon (Radman, 1975). The initial molecular signal that causes activation of the SOS response is believed to be the single-stranded DNA that forms during replication of damaged DNA (Sassanfar and Roberts, 1990). Recently, it was shown that a replication block in one strands triggers the functional uncoupling of the coordinated replication of the leading and lagging strands in vivo, thus producing extended single-stranded DNA regions mostly when the lesion resides in the leading strand (Pages and Fuchs, 2003). Single-stranded DNA is then converted into the 'sOS signal' via the formation of a RecA nucleofilament that facilitates autocleavage of the LexA repressor and consequently upregulates the transcription of at least 43 SOS genes (Fernandez De Henestrosa et al, 2000; Courcelle et al, 2001). Distinct cellular strategies usually referred to as tolerance mechanisms have evolved to cope with replication-blocking lesions. Among the tolerance mechanisms, translesion synthesis (TLS) involves copying the damage-containing template with the help of specialized DNA polymerases that transiently replace the replicative DNA polymerase (Cordonnier and Fuchs, 1999; Sutton and Walker, 2001; Goodman, 2002; Pages and Fuchs, 2002). Among the SOS-induced genes, three encode DNA polymerases (polB, dinB and umuDC), which have all recently been shown to be involved in lesion bypass in vivo (Napolitano et al, 2000; Wagner et al, 2002). Pol V, the major bypass polymerase (Reuven et al, 1999; Tang et al, 1999), is composed of one UmuC and a dimer of UmuD′ molecules, the RecA* catalyzed autocleaved form of UmuD (Burckhardt et al, 1988; Shinagawa et al, 1988). In addition, Pol II and Pol IV may also participate in TLS either alone or in combination with Pol V depending upon the nature of the lesion and its local sequence context (Napolitano et al, 2000; Wagner et al, 2002). The process of TLS, which is inevitably error-prone, is responsible for the majority of induced point mutations. In vivo, the process of TLS involves the recruitment of one or several specialized DNA polymerases (Napolitano et al, 2000; Bresson and Fuchs, 2002; Prakash and Prakash, 2002; Wagner et al, 2002) that will perform limited DNA synthesis in the vicinity of the damage, thus allowing the replicative polymerase to resume synthesis following bypass of the lesion. A key factor involved in these processes is the general replication processivity factor, the β-clamp, shown to interact with all E. coli DNA polymerases in vitro (Lopez de Saro et al, 2003) and in vivo (Dalrymple et al, 2001; Becherel et al, 2002; Lenne-Samuel et al, 2002). A key feature in the 'DNA polymerase switch model' (Cordonnier and Fuchs, 1999) is the definition of the transition points between specialized and replicative polymerases. The size of the patch made by the bypass DNA polymerase (TLS patch) needs to be optimized: it should be sufficiently long to allow successful elongation upon rebinding of the replicative polymerase and at the same time it should not be too long, as the bypass polymerases exhibit low fidelity and would consequently be responsible for the induction of untargeted mutations. In this paper we reconstituted the process of TLS using circular single-stranded DNA (ss-circular DNA) templates containing a single DNA lesion, the highly purified DNA polymerases from E. coli, namely the replicative Pol III holoenzyme (Pol III HE), Pol V and Pol II. The circular substrate used here allows stable loading of the necessary accessory factors, namely an ATP-activated RecA filament and the β-clamp. The present work highlights the key role of the β-clamp that is donated to Pol V by Pol III HE following its dissociation in the vicinity of the lesion site. The donated β-clamp specifically increases the processivity of Pol V, allowing it to synthesize, in a single binding event, a TLS patch long enough to escape Pol III-mediated exonuclease degradation. The minimal TLS assay described here recapitulates the major in vivo requirements of induced mutagenesis. Results Overall goals and strategy The overall goal of the present investigation is to define the position of the switches between replicative and specialized DNA polymerases during lesion bypass. For this purpose, E. coli was chosen as a model system, as the genetics of induced mutagenesis in this organism has been well documented over the years (Friedberg et al, 1995). From the data gathered in vivo, it is clear that in order to reconstitute the whole process of TLS in vitro, one needs to set up an assay that not only includes the polymerases themselves but also some of the essential accessory factors that play a key role in the bypass process. Indeed, in addition to the replicative polymerase (Pol III HE; for a recent review, see McHenry, 2003) and the major bypass polymerase (Pol V), genetic data on umuDC-dependent mutagenesis clearly involve both an activated RecA-single-stranded DNA filament and the β-clamp, the general replication processivity factor (Dutreix et al, 1989; Becherel et al, 2002; Goodman, 2002). Following unsuccessful attempts with linear oligonucleotide substrates, we found that long circular single-stranded templates offer a simple experimental system to set up a minimal TLS assay. Indeed, these circular templates support both the stable loading of the β-clamp and the formation of extended RecA filaments in the presence of ATP. The major objectives of the present work are (i) to define the position, with respect to the lesion site, of the switches between replicative and specialized DNA polymerases, (ii) to analyze the synthesis profiles when replicative and specialized polymerases are present either separately or together and (iii) to evaluate potential competition among polymerases during lesion bypass. We wanted to determine both the average length of the synthesis patch made by Pol V in a single binding event (defined here as the 'TLS patch') and the minimal distance from the primer terminus a lesion needs to be located to escape degradation by the proofreading function upon Pol III rebinding. Mixing replicative and specialized DNA polymerases: effect on lesion bypass profiles In an attempt to reconstitute TLS, we performed a series of experiments involving replicative and bypass polymerases, either alone or in combination (Figure 1 and Table I). We purified DNA polymerase III* (Pol III*) (see Materials and methods), the dimeric form of the core DNA polymerase III that also includes the γ complex (McHenry, 2003). In the presence of the β-clamp, Pol III* forms E. coli's replicative enzyme, Pol III HE (McHenry, 2003). Pol V and Pol II were extensively purified and characterized as described (Becherel and Fuchs, 2001; Fujii et al, 2004). These experiments involve RecA-coated single-stranded circular templates in the presence of ATP and primers located 6 or 8 nucleotides (nt) upstream (running start conditions) from a single G-AAF adduct within the NarI (Figure 1A and B) or the 3G sequence context (Figure 1C), respectively. In E. coli, the NarI context is a hot spot for −2 frameshift mutagenesis induced by G-AAF adducts (Fuchs et al, 1981; Koffel-Schwartz et al, 1984). Extensive in vitro and in vivo studies have established that G-AAF adducts bound to the underlined guanine (5′-GGCGCC-) can be bypassed either by Pol V or Pol II in an error-free or a −2 frameshift pathway, respectively (Figure 1) (Napolitano et al, 2000; Becherel and Fuchs, 2001; Fujii et al, 2004). The 3G context is a comparatively weaker −1 frameshift hot spot that requires Pol V for both error-free and frameshift bypass (Napolitano et al, 2000). Figure 1.Interplay between polymerases during lesion bypass. All experiments involve circular single-stranded templates (≈2.7 kb) containing a G-AAF adduct located either within the NarI sequence primed with L-6 (A, B) or within the 3G sequence primed with L-8 (C). Standard reaction mixtures containing 50 nM β, 2 μM RecA and 10 nM SSB (see Materials and methods) were preincubated with or without γ complex. When indicated, reactions were initiated by adding Pol III* (III) for 3 min at 30°C, followed by the addition of Pol II and/or Pol V for an additional 60 min. Incubations with Pol V alone were performed for 15 min. The concentrations of enzymes used are as follows: (A) Pol III*: 4 nM, Pol II: 4 nM, Pol V: 100 nM; (B) Pol III*: 62.7 nM, Pol II: 4 nM, Pol V: 100 nM; (C) Pol III*: 20 nM, Pol II: 4 nM, Pol V: 100 nM. The reaction mixtures were digested by EcoRI located 11 and 14 nt downstream of the lesion in the 3G and NarI contexts, respectively. The reaction products are analyzed by 10% denaturing PAGE. Download figure Download PowerPoint Table 1. Efficiency of translesion synthesis (derived from Figure 1) A B C 1 2 3 4 5 6 7 8 1 2 3 4 1 2 3 4 5 Sample Pol IIIa + − − + − − + + + + + + + − − + + Pol V − + + + − − − + − + − + − + + + − Pol II − − − − + + + + − − + + − − − − + γ complex − − + − − + − − − − − − − − + − − Final product (%) TLS0 0 <0.1a 20a 24 <0.1 <0.1 <0.1 21 0 11 <0.1 7.8 0 <0.1a 14 11 <0.1 TLS-1 <0.1 <0.1 <0.1 <0.1 0.5 19 4.2 3.7 <0.1 <0.1 0.3 0.2 1.7 <0.1 <0.1 1.5 2.0 TLS-2 8.2 <0.1 <0.1 7.0 5.5 16 36 34 5.5 5.9 36 32 <0.1 <0.1 <0.1 <0.1 40 nt) can be seen at all time points. These products result from a minor polymerase contamination, present in the γ complex preparation, that is stimulated by the β-clamp and inhibited by the G-AAF adduct. Download figure Download PowerPoint Table 2. Kinetic and processivity data of Pol V in the presence of the β-clamp Sample Average processivity (nt) Average velocity (nt/s) Maximum product length up to (nt) Nar0/−β 0.5–8 min 3 >0.10 12 Nar0/+β 0.5 min 8–9 ≈0.28 18 1 min 17–18 ≈0.29 31 2 min ≈25 — 55 4 min ≈25 — >100a 8 min ≈25 — >100a Nar3/+β 0.5 min 2–3 ≈0.083 13 1 min 10–11 ≈0.18 26 2 min ≈18 — 48 4 min ≈18 — >60b 8 min ≈18 — >60b These data are derived from the gel shown in Figure 2. Nar0 and Nar3 refer to undamaged and G-AAF-containing templates, respectively. Processivity data are determined by intensity-weighed averaging. a Products longer than >100 nt represent ≈9% of total products; a minor polymerase contamination in the γ complex preparation accounts for the low amount of long extension products (not seen in the presence of the adduct). b Products longer than >60 nt represent ≈1% of total products. No product is detected when Pol V is incubated with Nar3 in the absence of the β-clamp under single hit conditions (Figure 2). By comparing the progression of the enzyme on lesion-free and lesion-containing templates, the average delay for bypassing the G-AAF adduct with Pol V can be estimated to be in the order of 24 s (Figure 2). It should be noted that despite a broad distribution of patch sizes, that is, from 1 to ≈60 nt, most TLS patches (≈75%) exceed the minimal length of 5 nt beyond the lesion that is necessary for Pol III-mediated elongation (see below and model in Figure 5). Pol III HE efficiently resumes DNA synthesis on RecA-covered single-stranded DNA template Following the dissociation of Pol V after lesion bypass, as the template is covered with RecA, we wanted to investigate the capacity of Pol III HE to use an ATP-activated RecA nucleofilament as a substrate for DNA synthesis. For this purpose, a primed ss-circular DNA template coated with RecA was used as a substrate for Pol III HE. Primer elongation proceeds efficiently whether the template is naked or covered with stoichiometric quantities of RecA protein in the presence of ATP (Figure 3). Similarly, low (10 nM) or high (300 nM) concentrations of SSB do not affect the activity of Pol III HE (Figure 3). The template used in these experiments contains a lesion 37 nt downstream from the 3′-end of the primer that makes Pol III stop at L-1. Figure 3.Pol III HE efficiently replicates an ATP-activated RecA filament. A circular single-stranded template (≈2.7 kb) containing a G-AAF adduct located within the NarI sequence primed with L-37 is used as a substrate for Pol III HE elongation experiments in the presence or absence of RecA and SSB. The DNA template was preincubated with SSB and RecA at the indicated concentrations in the presence of β-clamp (50 nM) for 10 min. Elongation reactions were initiated by adding 8 nM Pol III* and terminated after 13 min. All reaction products were digested by EcoRI and analyzed by a 10% denaturing PAGE. M indicates DNA size markers. Download figure Download PowerPoint Defining the minimal size of the TLS patch that is required for efficient elongation by DNA Pol III HE For a series of common replication-blocking DNA lesions, we wanted to measure the effect of the position of the 3′-end of the primer, with respect to the lesion site, on the capacity of the replicative polymerase to bind and extend the nascent strand. Either linear oligonucleotides (130-mer) or circular single-stranded vectors (2700 nt) were used as substrates following annealing with a series of complementary primers. For the linear templates, the primer annealing site was centrally located leaving both 3′ and 5′ single-stranded template overhangs. The templates were preincubated with SSB, followed by the addition of Pol III* (McHenry, 2003) and β-clamp in the presence of a mixture of all four dNTPs. Under these conditions, if Pol III HE cannot elongate a primer given the distortion imposed by the lesion in the template strand, we will observe primer degradation by its associated exonuclease activity despite the presence of dNTPs. The relative efficiencies of polymerization (% pol) and degradation (% exo) for a given primer Ln (listed above each panel in Figure 4) were quantified as follows: % pol, the sum of band intensities above position Ln divided by the sum of all bands except Ln; % exo, the sum of band intensities below position Ln divided by the sum of all bands except Ln. Figure 4.Pol III HE and Pol II require a minimum of 4 or 5 base pairs beyond the lesion to resume efficient DNA synthesis. Substrates are either linear oligonucleotides or circular single-stranded plasmids onto which 5′-end-labeled primers are annealed. For linear oligonucleotides (130-mer), the lesion is located about halfway from both ends of the template (details in Materials and methods). Lesions are G-AAF (D–H), AP site (A), TT cyclobutane dimer (B) and TT (6-4) photoproduct (C). Each panel shows the reaction products using a series of primers a various length ranging from L-2 (24-mer) to positions up to L6 (32-mer). Primers are named according to the position their 3′-end anneals to the template with respect to the lesion site (i.e., the 3′-end nucleotide of primer L2 ends 2 nt beyond the lesion site that is referred to as L0). In (F, H), the G-AAF adduct located within the NarI frameshift hot spot forms a −2 frameshift intermediate (Fuchs et al, 1981; Burnouf et al, 1989); '−2 frameshift' primers are named as follows: primer Ln(−2) designates a primer that ends at position n (with respect to the lesion site) in the absence of misalignment. When a −2 nt misalignment intermediate forms, primer Ln(−2) ends at position n+2 as shown in the top of (F, H). All Pol III reactions (A–F) were carried out under the standard conditions containing 50 nM β-clamp and 300 nM SSB (see Materials and methods). Pol II reactions (G, H) did not contain the β-clamp. Reactions were preincubated (10 min at 30°C), initiated by adding 2 nM Pol III* (A–F) or 4 nM Pol II (G, H) and terminated after 15 min. The reaction products were analyzed by 10% denaturing PAGE. (E) G-AAF in the NarI context is located in a circular template to evaluate the potential effect of circular versus linear templates. Reaction products in (E) were digested with EcoRI before electrophoresis. P and M indicate DNA size markers. For each reaction, the efficiencies of polymerization (% pol) and degradation (% exo) (listed above each panel) were calculated as follows: % pol, the sum of band intensities above position Ln divided by the sum of all bands except Ln; % exo, the sum of band intensities below position Ln divided by the sum of all bands except Ln. Download figure Download PowerPoint Figure 5.An integrated view of TLS. (A) Pol III* associated with its processivity factor the β-clamp encounters a noncoding lesion in the template and stops. A RecA filament forms on the single-stranded DNA region downstream the lesion site. This RecA