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. 2014 Oct 10;289(41):28388-98.
doi: 10.1074/jbc.M114.587907. Epub 2014 Aug 19.

Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB

Sankalp Gupta  1 , Joseph T P Yeeles  1 , Kenneth J Marians  2
Affiliations

Regression of replication forks stalled by leading-strand template damage: II. Regression by RecA is inhibited by SSB

Sankalp Gupta et al. J Biol Chem. .

Abstract

Stalled replication forks are sites of chromosome breakage and the formation of toxic recombination intermediates that undermine genomic stability. Thus, replication fork repair and reactivation are essential processes. Among the many models of replication fork reactivation is one that invokes fork regression catalyzed by the strand exchange protein RecA as an intermediate in the processing of the stalled fork. We have investigated the replication fork regression activity of RecA using a reconstituted DNA replication system where the replisome is stalled by collision with leading-strand template damage. We find that RecA is unable to regress the stalled fork in the presence of the replisome and SSB. If the replication proteins are removed from the stalled fork, RecA will catalyze net regression as long as the Okazaki fragments are sealed. RecA-generated Holliday junctions can be detected by RuvC cleavage, although this is not a robust reaction. On the other hand, extensive branch migration by RecA, where a completely unwound product consisting of the paired nascent leading and lagging strands is produced, is observed under conditions where RuvC activity is suppressed. This branch migration reaction is inhibited by SSB, possibly accounting for the failure of RecA to generate products in the presence of the replication proteins. Interestingly, we find that the RecA-RuvC reaction is supported to differing extents, depending on the template damage; templates carrying a cyclopyrimidine dimer elicit more RecA-RuvC product than those carrying a synthetic abasic site. This difference could be ascribed to a higher affinity of RecA binding to DNAs carrying a thymidine dimer than to those with an abasic site.

Keywords: DNA Enzyme; DNA Recombination; DNA Repair; DNA Replication; Genomic Instability.

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Figures

FIGURE 1.
FIGURE 1.
RecA does not affect DNA replication directly. Replication reaction mixtures (20 μl) were as described under “Experimental Procedures” except that the SSB concentration (monomer) was as indicated. RecA (3 μm) was added where indicated at the same time as the EcoRI and [α-32P]dATP. Aliquots (5 μl) were removed at the indicated times post-EcoRI addition, and the DNA replication reaction was terminated by the addition of two volumes of STOP buffer. After an additional 10 min of incubation to digest the DNA products with PvuI, EDTA was added, and the reaction products were analyzed by either neutral gel electrophoresis (A) or denaturing alkaline gel electrophoresis (B). SF, stalled forks; FL, the full-length EcoRI-PvuI DNA product; NDD, nascent DNA duplex; RS, restart products; OF, Okazaki fragments.
FIGURE 2.
FIGURE 2.
Formation of a RFR product by RecA in the presence of the replisome is stimulated by sealing the Okazaki fragments. Stalled forks formed in either the absence (without Ligase; A) or presence (with Ligase; B) of DNA polymerase I, RNase H, and DNA ligase to seal the Okazaki fragments were incubated in standard RFR reaction mixtures (14) containing 10 nm RuvC, 10 nm RecG, and 0.75–6 μm RecA (in increments of 2-fold; lanes 4–7), as indicated, for 20 min at 37 °C. DNA products were analyzed by native gel electrophoresis. C, quantification of the “with ligase” experiment (average of two experiments). Error bars, S.D.
FIGURE 3.
FIGURE 3.
Formation of RecA RFR products with deproteinized stalled forks requires sealed Okazaki fragments. Standard RFR reaction mixtures with deproteinized stalled forks formed either in the absence (without Ligase) or presence (with Ligase) of DNA polymerase I, RNase H, and DNA ligase, containing RecG (10 nm), RuvC (10 nm), or RecA (3 μm), as indicated, were incubated for 20 min at 37 °C. DNA products were analyzed by native gel electrophoresis. The histogram shows quantification of the amount of either CP2 or NDD produced (average of three experiments). Error bars, S.D.
FIGURE 4.
FIGURE 4.
Schematic of the RecA RFR reaction. i, the stalled fork; ii, lagging-strand and leading-strand sisters aligned; iii, reorientation of the lagging-strand sister; iv, recombinant joint molecule formed by invasion of the nascent leading strand from the leading-strand sister into the lagging-strand sister; v, unsealed Okazaki fragments and gaps between them could prevent branch migration from proceeding. When the Okazaki fragments are sealed (v), this possibility is eliminated, and the branch migration reaction should proceed. The arrow denotes the direction of branch migration. Black lines, lagging-strand template; green lines, leading-strand template; blue lines, nascent lagging-strand DNA; red lines, nascent leading-strand DNA.
FIGURE 5.
FIGURE 5.
SSB stimulates RuvC cleavage of deproteinized stalled forks. A, standard RFR reaction mixtures with deproteinized stalled forks formed in the presence of DNA polymerase I, RNase H, and DNA ligase containing 0.25–2 μm SSB (increasing in 2-fold increments in lanes 5–8) were incubated at 37 °C for 5 min. RuvC and RecA were then added as indicated to 10 nm and 3 μm, respectively, and the incubation continued for 20 min at 37 °C. The DNA products were analyzed by native gel electrophoresis. B, quantification of the amount of CP2 and NDD formed (as a fraction of the total DNA products). The mean and S.D. (error bars) from three experiments is shown. A representative gel is shown in A. C, SSB inhibits RuvC cleavage of a model stalled fork, where the nascent lagging strand is ahead of the nascent leading strand. A model stalled fork was formed, as described under “Experimental Procedures,” that had a duplex lagging-strand sister arm and a single-stranded leading-strand sister arm. Reaction mixtures containing the model stalled fork with the nascent lagging-strand labeled at the 5′-end with 32P, 1 μm SSB, and 10 nm RuvC, as indicated, were incubated at 37 °C for 10 min. The reactions were terminated by the addition of EDTA to 30 mm, and the products were analyzed by electrophoresis through an 8% polyacrylamide gel (29:1, acrylamide/bisacrylamide).
FIGURE 6.
FIGURE 6.
RecA branch migrates the nascent strands off of the template strands of deproteinized stalled forks at lower magnesium concentrations. A, RecA generates a RFR product in the absence of RuvC. Standard RFR reaction mixtures at 3 mm Mg(OAc)2 either in the presence or absence of 10 nm RuvC, as indicated, and in the presence of RecA (from 0.75 to 6 μm, increasing by 2-fold in lanes 2–5 and 7–10) were incubated for 20 min at 37 °C, and the DNA products were analyzed by native gel electrophoresis. B, dependence of RuvC cleavage on Mg2+ concentration. RuvC was incubated with a synthetic HJ (HJ1 (28)) as described under “Experimental Procedures” at the indicated concentrations of Mg(OAc)2. Cleavage products were analyzed by electrophoresis through a 8% polyacrylamide gel. C and D, the RecA RFR product is a nascent strand DNA duplex. Standard RFR reaction mixtures at 3 mm Mg(OAc)2 containing 3 μm RecA, RuvAB (40–120 nm), or no RFR proteins were incubated for 20 min at 37 °C. Either DpnI (1 unit) (C) or MboI (0.1 unit) (D) was then added, and the incubation continued for 10 min. DNA products were then analyzed by native agarose gel electrophoresis. E and F, SSB inhibits formation of the RecA RFR product. Standard RFR reaction mixtures at 3 mm Mg(OAc)2 containing 3 μm RecA and either SSB (E) or SSB W54S (F) (25–500 nm, increasing by 2-fold in lanes 3–8), as indicated, were incubated for 20 min at 37 °C, and the DNA products were analyzed by native gel electrophoresis.
FIGURE 7.
FIGURE 7.
RecA does not interfere with RuvAB RFR. A, comparison of the kinetics of RecA and RuvAB RFR. RFR reaction mixtures (100 μl) containing deproteinized SFs and either 3 μm RecA or RuvAB (40–120 nm) were incubated at 37 °C. Aliquots (15 μl) were removed at the indicated times, the reactions were stopped by the addition of 30 mm EDTA, and the products were analyzed by native gel electrophoresis. B, RecA does not interfere with RuvAB RFR. RFR reaction mixtures containing the replisome and SSB, as described in the accompanying article (14), containing RecA (3 μm), RecG (10 nm), and RuvAB (40–120 nm), as indicated, were incubated for 10 min at 37 °C, and the products were analyzed by native gel electrophoresis.
FIGURE 8.
FIGURE 8.
CPD templates support a greater extent of RecA RFR than THF templates. A and B, standard RFR reaction mixtures with deproteinized stalled forks formed from either the 225CPD template (the standard template) or the 5903THF template (a template with a THF in the same position as the CPD on the 225 template) (A) or the 119CPD and 119THF templates (a pair of templates where the CPD and THF are in identical positions, 3.27 kbp from the PvuI site) (B) at 10 mm Mg(OAc)2 containing 10 nm RuvC and 3 μm RecA, as indicated, were incubated for 20 min at 37 °C, and the DNA products were analyzed by native gel electrophoresis. C, quantification of the ratio of product formation, RecA-RuvC/RuvC, on the various template DNAs. D and E, SSB-stimulated RuvC cleavage is identical on the CPD and THF templates. Standard RFR reaction mixtures with deproteinized stalled forks formed from either the 225CPD template or the 5903THF template at 10 mm Mg(OAc)2 containing 10 nm RuvC, 3 μm RecA, and 1 μm SSB, as indicated, were incubated for 20 min at 37 °C, and the DNA products were analyzed by native gel electrophoresis. F, quantification of the amount of either CP2 and NDD formed in the experiments shown in panels D and E. The mean and S.D. (error bars) of three experiments is shown in C and F. Representative gels are shown in A, B, D, and E.
FIGURE 9.
FIGURE 9.
RecA displays preferential binding to model oligonucleotide substrates carrying a CPD compared with a THF. A, schematic of the oligonucleotide substrates. DNA binding reaction mixtures containing either the CPD or THF oligonucleotide substrates at 10 mm Mg(OAc)2 (B and C, respectively) and increasing amounts of RecA (no RecA, and 0.04–2.5 μm RecA, increasing by a factor of 2-fold from left to right) were incubated at 37 °C for 10 min and then analyzed by electrophoresis through native polyacrylamide gels as described under “Experimental Procedures.” D, DNA binding curves. Shown are the mean and S.D. (error bars) from three experiments. Representative gels are shown. Sub, substrate.
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