E. coli DNA replication in the absence of free β clamps
2011; Springer Nature; Volume: 30; Issue: 9 Linguagem: Inglês
10.1038/emboj.2011.84
ISSN1460-2075
AutoresNathan A. Tanner, Gökhan Tolun, Joseph J. Loparo, Slobodan Jergic, Jack D. Griffith, Nicholas E. Dixon, Antoine M. van Oijen,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoArticle25 March 2011free access E. coli DNA replication in the absence of free β clamps Nathan A Tanner Nathan A Tanner Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Zernike Institute for Advanced Materials, University of Groningen, Groningen, NetherlandsPresent address: New England Biolabs, Ipswich, MA 01938, USA Search for more papers by this author Gökhan Tolun Gökhan Tolun Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Joseph J Loparo Joseph J Loparo Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Slobodan Jergic Slobodan Jergic School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia Search for more papers by this author Jack D Griffith Jack D Griffith Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Nicholas E Dixon Nicholas E Dixon School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia Search for more papers by this author Antoine M van Oijen Corresponding Author Antoine M van Oijen Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Zernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands Search for more papers by this author Nathan A Tanner Nathan A Tanner Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Zernike Institute for Advanced Materials, University of Groningen, Groningen, NetherlandsPresent address: New England Biolabs, Ipswich, MA 01938, USA Search for more papers by this author Gökhan Tolun Gökhan Tolun Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Joseph J Loparo Joseph J Loparo Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Slobodan Jergic Slobodan Jergic School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia Search for more papers by this author Jack D Griffith Jack D Griffith Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA Search for more papers by this author Nicholas E Dixon Nicholas E Dixon School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia Search for more papers by this author Antoine M van Oijen Corresponding Author Antoine M van Oijen Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA Zernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands Search for more papers by this author Author Information Nathan A Tanner1,2, Gökhan Tolun3, Joseph J Loparo1, Slobodan Jergic4, Jack D Griffith3, Nicholas E Dixon4 and Antoine M van Oijen 1,2 1Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA 2Zernike Institute for Advanced Materials, University of Groningen, Groningen, Netherlands 3Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA 4School of Chemistry, University of Wollongong, Wollongong, New South Wales, Australia *Corresponding author. Single-Molecule Biophysics, Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 4, Groningen 9747 AG, Netherlands. Tel.: +31 050 363 9883; Fax: +31 050 363 3660; E-mail: [email protected] The EMBO Journal (2011)30:1830-1840https://doi.org/10.1038/emboj.2011.84 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info During DNA replication, repetitive synthesis of discrete Okazaki fragments requires mechanisms that guarantee DNA polymerase, clamp, and primase proteins are present for every cycle. In Escherichia coli, this process proceeds through transfer of the lagging-strand polymerase from the β sliding clamp left at a completed Okazaki fragment to a clamp assembled on a new RNA primer. These lagging-strand clamps are thought to be bound by the replisome from solution and loaded a new for every fragment. Here, we discuss a surprising, alternative lagging-strand synthesis mechanism: efficient replication in the absence of any clamps other than those assembled with the replisome. Using single-molecule experiments, we show that replication complexes pre-assembled on DNA support synthesis of multiple Okazaki fragments in the absence of excess β clamps. The processivity of these replisomes, but not the number of synthesized Okazaki fragments, is dependent on the frequency of RNA-primer synthesis. These results broaden our understanding of lagging-strand synthesis and emphasize the stability of the replisome to continue synthesis without new clamps. Introduction The replicative DNA polymerase III of Escherichia coli is a heterotrimer of the polymerase subunit α, the exonuclease ε, and a stabilizing accessory protein θ (McHenry and Crow, 1979; Kelman and O'Donnell, 1995; Johnson and O'Donnell, 2005). This complex, termed the 'core' polymerase, is a relatively distributive enzyme, capable of only 10–20 nucleotide (nt) additions at ∼10 nt s−1 (Fay et al, 1981; Maki et al, 1985). These numbers are dramatically less than is required to replicate the 4.6 mega-basepair (Mb) genome in its 40-min duplication time. To a large extent, polymerase stimulation is conferred by core association with the homodimeric processivity clamp β, which encircles double-stranded DNA (dsDNA) and, through interaction with α, tethers the polymerase to a primer-template junction (Fay et al, 1981; Georgescu et al, 2008). In complex with β, polymerase activity increases to a rate of 350–500 nt s−1 and a processivity of 1–2 kilo-basepairs (kb) (Tanner et al, 2008). The loading of β onto dsDNA is achieved by a multiprotein clamp-loader complex which contains three copies of either a truncated (γ) or a full-length (τ) version of the dnaX gene product, which hydrolyses ATP to open and load β and, with the C-terminal domains of τ, binds α and the helicase DnaB; one copy each of δ and δ′, which provide structural support for β opening and loading; and the accessory proteins χ and ψ, which interact with single-stranded DNA-binding protein (SSB) and primase DnaG and contribute to regulating the primer handoff cycle (Glover and McHenry, 1998; Jeruzalmi et al, 2001; Johnson and O'Donnell, 2005; Bloom, 2006). The clamp loader and its associated core polymerases form the Pol III* complex, and addition of the β clamps to Pol III* forms the holoenzyme (Pol III HE). Since the first analysis of cell lysates, and as depicted in models, the clamp-loader complex has been thought to include one copy of γ and two of τ, one each for leading- and lagging-strand polymerases (McHenry and Kornberg, 1977; Onrust et al, 1995). However, recent identification of a τ3-(αεθ)3 replisome and in vivo stoichiometry measurements have suggested that the replisome contains three polymerases (McInerney et al, 2007; Reyes-Lamothe et al, 2010). The presence of the additional core has been proposed to have a role in rescuing stalled replisomes or facilitating the lagging-strand cycle for efficient fork progression. During synthesis of the lagging strand, the active polymerase must cycle from one Okazaki fragment to the next. The polymerase may complete the Okazaki fragment and release upon reaching the 5′ end of the previous fragment ('collision' release, Stukenberg et al, 1994; Georgescu et al, 2009), or release before completion of the Okazaki fragment due to a biochemical signal, for example, primer synthesis or other lagging-strand event ('signalling' release, Li and Marians, 2000; McInerney and O'Donnell, 2004). In either case, the lagging-strand polymerase should disengage from one β clamp and reassociate with another loaded on the new RNA primer. Indeed, biochemical studies demonstrate that a Pol III core quickly releases from β after completing a fragment, and that this core will recycle to a new primed, β-loaded substrate (O'Donnell, 1987; Stukenberg et al, 1994; Georgescu et al, 2009). Traditionally, the β clamp to be loaded on the nascent primer is thought to associate with the replisome from solution. The clamp on the finished fragment is left behind after core dissociation, for binding other enzymes, such as those involved in Okazaki fragment maturation (Yuzhakov et al, 1996; López de Saro and O'Donnell, 2001). Considering the >10-fold difference between the number of β dimers in the cell (∼300; Burgers et al, 1981) and the number of Okazaki fragments made during chromosome duplication (∼4000), it is clear that clamps must be reused to allow rapid duplication of the genome. Given that β loaded on circular dsDNA is stable for >60 min without dissociating, the clamps must be actively unloaded (Yao et al, 1996). Studies have demonstrated that the forms of the clamp-loader complex and free δ are effective clamp unloaders, and this unloading can allow clamps to be reused after subsequent diffusion to the replisome (Naktinis et al, 1996; Yao et al, 1996; Leu et al, 2000). Thus, our current model of the lagging-strand cycle includes a lagging-strand polymerase releasing from a β clamp and associating with a 'new' clamp that has been loaded from solution. However, this mechanism is not completely elucidated; many of the details of clamp unloading and reuse have either not been addressed in the context of coordinated replication by intact replisomes or have been studied using ensemble techniques that can overlook rare or transient events. Here, we use single-molecule fluorescence and electron microscopy to examine replication by complexes unable to complete the traditional lagging-strand clamp-loading cycle. Our understanding of DNA replication mechanisms is largely based on years of pioneering biochemical experiments that have elucidated the functions and interactions of the various replisome components. Though extremely powerful, traditional ensemble methods inherently obscure rare events, intermediate steps, and even alternate pathways. In recent years, single-molecule methods have been developed to allow the observation of these types of events and have emerged as a powerful complement to traditional bulk-phase techniques, enabling the characterization of primase-induced leading-strand synthesis stalling (Lee et al, 2006; Tanner et al, 2008), dynamic control of lagging-strand trombone loops (Hamdan et al, 2009), priming loops (Manosas et al, 2009; Pandey et al, 2009) and many other facets of replisome function (reviewed in Hamdan and van Oijen (2010) and van Oijen and Loparo (2010)). We recently demonstrated a technique for visualizing coordinated leading- and lagging-strand synthesis with single-molecule fluorescence microscopy (Tanner et al, 2009). By tethering rolling-circle DNA substrates (Mok and Marians, 1987) to the surface of a microscope-mounted flow chamber, we can observe the growing dsDNA product (lagging strand) of a synthesis reaction with a fluorescent DNA stain. This technique has allowed measurement of the rate and processivity of coordinated synthesis by single replisomes (Tanner et al, 2009; Yao et al, 2009). The real-time visualization of individual replication products allows full characterization of the dynamics of the replication reaction, including the observation of heterogeneity in rates. Furthermore, product lengths of up to hundreds of kb can be measured, far beyond the resolving power of traditional gel electrophoresis measurements. Here, we adapt the single-molecule rolling-circle assay to facilitate pre-assembly of stable, stalled replisome complexes followed by initiation of synthesis in the absence of free β clamps. Surprisingly, we observed processive synthesis without β in solution, a result that requires either the lagging-strand polymerase to function without a β clamp or, more consistent with our data, recycling of the β clamp from the end of an Okazaki fragment to a nascent RNA primer. Products synthesized without free clamps were reduced in length and rate as compared with those synthesized under normal conditions, but still contained multiple Okazaki fragments. The absence of free β rendered the processivity of the replisome sensitive to DnaG primase concentration; reducing the concentration of DnaG increased the average product length. We demonstrate that in the absence of free β, the product length increased by the same factor as the spacing of RNA primers, which were still produced and extended in the absence of free β. Together, and in contrast with established dogma, these data demonstrate that the production of multiple Okazaki fragments on the lagging strand can be supported even without the replisome recruiting and loading additional clamps from solution. Results Pre-assembled replisomes produce long replication products We have previously shown observation of rolling-circle replication products with single-molecule fluorescence microscopy (Tanner et al, 2009). These products are the result of leading-strand synthesis producing a single-stranded 'tail', which is converted to dsDNA by coupling of leading- with lagging-strand synthesis (Figure 1). As the dye (SYTOX Orange) stains only dsDNA, we exclusively observe the products of coupled synthesis. These experiments were performed in the continuous presence of all replisome components, and we observed a product length of 85.3±6.1 kb (fit decay constant±s.e.m.). Yao et al (2009) showed that replisomes could be pre-assembled and we adapt their method here. Replisomes were assembled at a primer terminus in a solution containing first DNA, DnaB, and ATP, followed by addition of Pol III HE components and two of the four dNTPs. This solution was diluted and flowed into the chamber, followed by washing with buffer containing the two dNTPs but no replication proteins. Coupled leading- and lagging-strand replication could be observed on introduction of a replication solution containing only DnaG, β, SSB, and all rNTPs and dNTPs. In the pre-assembly reaction, we measured a processivity of 72.5±3.5 kb (Figures 1 and 2). This observation confirms that both Pol III* and DnaB are efficiently retained at the replication fork (Onrust et al, 1995; Yuzhakov et al, 1996; Johnson and O'Donnell, 2005; Yao et al, 2009). To ensure that the pre-assembly reaction indeed resulted in processive, coordinated leading- and lagging-strand replication by single replisomes, we carried out various control experiments. In particular, omission of only DnaG primase or using exclusively dNTPs in the second-stage replication solution (i.e., with no DnaG, β, or SSB; only pre-assembled Pol III HE components+DnaB) resulted in no detectable products. A reaction with a τ1γ2δδ′χψ (instead of τ2/3γ1/0δδ′χψ) clamp loader, capable of assembling only one core polymerase in the replisome, yielded no dsDNA products, indicating that a coupled lagging-strand polymerase is required to observe long products. Furthermore, when including SYTOX during the entire reaction including the pre-assembly step, we did not observe product formation until introduction of the replication solution containing all NTPs, DnaG, and SSB; during the pre-assembly and wash steps no synthesis was observed. By adding SYTOX only to the replication solution (no stain in pre-assembly or wash mixtures) we can visualize the entrance time of all NTPs, DnaG, etc. into the flow channel, and indeed, product growth occurred directly upon introduction of the SYTOX-containing solution in both β-containing and β-free conditions. These observations clearly establish that the pre-assembled replication complexes were responsible for the observed DNA synthesis. Figure 1.Visualizing replication products of pre-assembled replisomes. (A) Schematic of pre-assembly reaction. DnaB, Pol III HE components, ATP and two dNTPs are incubated with a pre-filled 5′-biotinylated rolling-circle substrate, which is coupled to a streptavidin-coated coverslip. After removal of free proteins, replication is initiated by addition of all r/dNTPs, SSB, DnaG, and ±β. (B, C) SYTOX staining of dsDNA in situ shows replicated products, with unextended substrates visible as small spots. Regions from fields of view after the full reaction performed with (B) and without (C) free β. Scale bars represent the length of a 10-kb dsDNA product. (D, E) Example molecules from real-time experiments are shown. Kymographs of individual replicating DNA molecules are shown from experiments with (D) and without (E) β in solution. The replication rate can be determined by fitting the end position of the growing molecule with linear regression as described in Tanner et al (2009). Shown molecules' average rates are 661 bp s−1 (D) and 210 bp s−1 (E); note the different length scales on the ordinates. Download figure Download PowerPoint Figure 2.Product distributions from pre-assembled replisomes. (A) Product lengths from reactions with the τ3-clamp loader. Inset shows values from single-exponential decay constant±s.e. N indicates the number of molecules observed. Full reaction (+β2, 300 nM DnaG): 72.5±3.5 kb, N=356; (−β2, 300 nM DnaG): 16.6±1.7 kb, N=366; (−β2, 30 nM DnaG): 43.5±3.1 kb, N=203. (B) Products from reactions with the τ2γ1-clamp loader. Full reaction (+β2, 300 nM DnaG): 40.2±2.7 kb, N=210; (−β2, 300 nM DnaG): 12.7±1.1 kb, N=249; (−β2, 30 nM DnaG): 33.8±3.4 kb, N=85. Download figure Download PowerPoint Absence of free β does not preclude synthesis To test whether the β clamps assembled with the replisome were sufficient for coordinated synthesis, we carried out the pre-assembly experiment without any β in the replication reaction, with only the small amount from the Pol III HE component–DnaB pre-assembly mixture. By extensive washing of the flow cell after introduction of the assembled complexes (∼30 chamber volumes), we removed any free proteins, leaving only the β that had been loaded onto the leading-strand primer-template and any β associated with the clamp loader or polymerase core. Surprisingly, we observed replication products in the absence of free β which were reduced in length (16.6±1.7 kb versus 72.5±3.5 kb; Figures 1C and 2A), but still much longer than a single Okazaki fragment (1–2 kb; Wu et al, 1992). Though resulting in processive replication, the efficiency of the reaction was notably decreased in the absence of free β clamps. In the full reaction, we observed 20–50 long products per 100 DNA molecules. With no free β in the reaction, this efficiency decreased nearly an order of magnitude, to 5–10 products per 100 DNA molecules. The decreased processivity and efficiency of replication may explain why this reaction had not been observed previously, specifically in similar single-molecule experiments limited to observing longer DNA products (Yao et al, 2009; Georgescu et al, 2010) and ensemble experiments less sensitive to short, inefficient replication (Yuzhakov et al, 1996). β is efficiently removed from the flow chamber To demonstrate that no unbound β remained to contribute to synthesis, we extended the washing volume to 250 chamber volumes (using an increased rate of flow, with a wash time of 5–10 min). Not surprisingly, this caused a reduction in the number of products observed ( 30 Cy5 molecules (saturation reached at ∼65 Cy5) and resulted in dramatically larger intensity decreases than the β distributions. These data show that our replisome-loaded DNA substrates were not filled with additional β molecules. Our observed low level of β loading could result from not satisfying conditions previously determined for loading of multiple βs, namely the presence of SSB on DNA and both a 5′ and 3′ primer end (Bloom et al, 1996). Also, the first step in our reaction was loading of DnaB at the fork to facilitate replisome-specific complex assembly, a step that may have prevented extraneous clamp loading. Rate of replication decreases in the absence of excess β clamps As described above, both the efficiency and processivity of replication decreased significantly without free β in the replication mixture. With our experimental design, we can also measure rates of replication by single replisomes. By including SYTOX and imaging the replication reaction in real time, we measured the rate of coordinated replication by generating a trajectory of the end point of a growing molecule (Tanner et al, 2009). Using the pre-assembly reaction followed by replication in the presence of β, we measured a rate of 460±41 bp s−1, similar to the rate of a reaction with all components present (both at 37°C). However, in the absence of free β, the replication rate of a pre-assembled replisome dropped to 228±27 bp s−1 (examples are shown in Figure 1D and E). A drop in rate would be expected if a lagging-strand core polymerase was extending the RNA primer, though this decrease would be expected to be much greater than the two-fold change we observe (Fay et al, 1981). A possible explanation for the two-fold rate decrease would be introduction of a slow intermediate step into the replication mechanism: the need to recycle a β clamp directly from the 3′ end of an Okazaki fragment to a nascent primer for the subsequent fragment synthesis. This would effectively introduce a pause in the replication trajectory at the beginning of each new Okazaki fragment, or every 1–2 kb (with 300 nM DnaG). However, the growing DNA end experiences zero stretching force under flow, leading to fluctuations in position that are much beyond the resolution necessary to confidently observe pauses during clamp recycling. Indeed, pauses of duration similar to Okazaki fragment synthesis (2–4 s) would result in an overall halving of the net rate of DNA synthesis. Free clamp-absent product length is sensitive to primase concentration The results described above unexpectedly suggest that the replisome can carry out lagging-strand synthesis without binding and loading a new β clamp. One potential mechanism for overcoming this problem would be the ability to directly reuse a clamp from the end of a completed Okazaki fragment to a new RNA primer. Such a mechanism is expected to have a certain probability of success per Okazaki fragment synthesized. If this idea is correct, the total processivity of the replisome with pre-assembled β should be determined not by the total amount of DNA synthesized, but by the total number of Okazaki fragments produced. To test this hypothesis, we reduced the concentration of DnaG primase in the reaction from 300 to 30 nM. This reduction has been shown to affect the frequency of DnaG-induced leading-strand synthesis halting (Tanner et al, 2008) and increase the length of Okazaki fragments due to similar reduction in priming frequency (Tougu and Marians, 1996). Here, using a clamp loader with three τ domains (τ3δδ′χψ), we observed that products at low DnaG concentration have a length of 43.5±3.1 kb, which represents a 2.6-fold increase in length compared with the products obtained with a high DnaG concentration (Figure 2A). Reducing the DnaG concentration had no effect on the replication rate, measured to be 239±26 bp s−1 with 30 nM DnaG, which is essentially identical to 228±27 bp s−1 observed with 300 nM DnaG. If the lagging-strand polymerase was synthesizing Okazaki fragments without being bound to a β clamp, it would likely not display sensitivity to primase concentration, as its increased likelihood of dissociation from the primer end affects processivity throughout the synthesis event rather than only upon RNA-primer handoff. This discrepancy suggests the clamp recycling mechanism is the more likely explanation for this observation. We next sought to test if the replication was facilitated by the presence of the third polymerase in the τ3 replisome. Using a clamp loader with two τ proteins (τ2γ1δδ′χψ), with replication observed in the absence of free β, gave products of 12.7±1.1 kb with 300 nM DnaG and 33.8±3.4 kb with 30 nM DnaG (Figure 2B). The measured length increase is a factor of 2.6, identical to the τ3 results. However, these lengths are only ∼75% of their τ3 counterparts, a result that agrees with an overall reduction in synthesis in τ2- versus τ3-mediated replication (McInerney et al, 2007). Indeed, when done with β in solution, the τ2 reaction gave a processivity of 40.2±2.7 kb, reduced from 72.5±3.5 kb in the τ3 reaction. Notably, we observed no pro
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