Yeast Rad5 protein required for postreplication repair has a DNA helicase activity specific for replication fork regression

Abstract

Lesions in the template DNA strand block the progression of the replication fork. In the yeast Saccharomyces cerevisiae, replication through DNA lesions is mediated by different Rad6-Rad18-dependent means, which include translesion synthesis and a Rad5-dependent postreplicational repair pathway that repairs the discontinuities that form in the DNA synthesized from damaged templates. Although translesion synthesis is well characterized, little is known about the mechanisms that modulate Rad5-dependent postreplicational repair. Here we show that yeast Rad5 has a DNA helicase activity that is specialized for replication fork regression. On model replication fork structures, Rad5 concertedly unwinds and anneals the nascent and the parental strands without exposing extended single-stranded regions. These observations provide insight into the mechanism of postreplicational repair in which Rad5 action promotes template switching for error-free damage bypass.

Figure 1

Stimulation of Rad5 ATPase by Various DNA Structures (A) (Aa) Possible DNA structures that can form by the annealing of 50 nucleotide-long, single-stranded oligonucleotides containing 25 dinucleotide repeats of dAdC or dGdT. (Ab) Schematic representation of defined DNA substrates used for Rad5 ATPase assays shown in (D). (B) DNA-dependent ATPase activity of Rad5. Forty nanomolar Rad5 was incubated with 1 mM [γ-³²P]ATP in the absence or presence of 100 nM single-stranded dAdC₍₂₅₎, single-stranded dGdT₍₂₅₎, or a mixture of these oligonucleotides at 37°C for 30 min. (C) Graphical representation of ATPase activity of Rad5 as the function of DNA concentration. ATPase activity of Rad5 (40 nM) was assayed at various concentrations of DNA (0–100 nM) as described in (B). (D) Kinetics of Rad5 (40 nM) ATPase activity in the presence of Y fork, heterologous fork, and X12 four-way junction-containing DNAs (10 nM). In (C) and (D), the mean of ATPase activity and standard deviation were calculated from three independent experiments.

Figure 2

Rad5 Possesses a DNA Helicase Activity Specific for Fork Reversal The DNA substrates in the gel are indicated by arrowheads, while the positions of some of the possible products are shown by arrows. The 3′ ends of oligonucleotides are indicated by half arrows, and the positions of 5′ ³²P labels are marked with asterisks. (A) Possible outcomes of DNA helicase action on a model replication fork substrate. (B) Replication fork regression activity of Rad5 tested on heterologous fork (HetF) and homologous fork (HomF) model substrates. We note that the formations of double-stranded products indicate fork processing through the d and d′ pathways shown in (A). (C) Homologous replication fork substrate labeled on both nascent strands. (D) Kinetics of Rad5 activity. Processing of homologous fork (HomF 30/30) (0.5 nM) was examined at different times in the presence of 25 nM Rad5. (E) Rad5 helicase activity requires ATP hydrolysis. Cofactor dependence was tested with 5 mM of various ribonucleotides in the presence of 5 mM MgCl₂, except in lane 5, where 10 mM EDTA was used instead of MgCl₂. (F) Rad5 ATPase mutant protein has no helicase activity. Rad5 DE refers to mutant Rad5 generated by changing the active-site DE residues at positions 681 and 682 to AA. (G) Rad5 can migrate a moveable four-way junction. The X0 junction is static while the core of the X12 junction is movable, which was flanked with heterologies that are 19 to 20 nucleotides long at the end of each arm.

Figure 3

Rad5 Can Regress Plasmid-Sized Model Forks (A) Schematic representation of the joint DNA substrate (pG46B′/pG68AXh) and the outcome of its Rad5-mediated regression. Letters A, H, R, X, S, F, Y, N, and Xh refer to restriction endonuclease sites AvrII, BamHI, EcoRI, BsaXI, SapI, AflIII, BseYI, AlwNI, and XhoI, respectively. The positions of 5′ ³²P labels on the “lagging strand” are marked with asterisk. (B) The extent of Rad5-dependent fork regression. The restriction enzyme site transfer to the regressed arm by Rad5 was followed in the presence of 5 mM ATP/Mg. The positions of the various restriction products generated by digestion of the regressed fork are indicated. Reaction with 5 mM AMP-PNP/Mg shows the background level of spontaneous regression. (C) Fork regression by Rad5 is progressive. Fork regression by Rad5 was compared on two joint DNA substrates containing either no heterology, or in which a 30 base pair sequence heterology was introduced at the SapI site, shown by arrowhead, of pG68A (named pG68 SapI[Het]). We note that the regression beyond the heterology was blocked, as revealed by the absence of F-, Y-, and N-specific bands. Reaction with gapped DNA (pG46B′) shows background due to nicking at the gap. (D) Fork reversal is not affected by E. coli ssDNA-binding protein. Regression through the BamHI site was monitored at various Rad5 concentrations (0–80 nM) in the presence or absence of E. coli SSB protein (460 nM).

Figure 4

Model for the Role of Rad5 in Lesion Bypass by Template Switching If replication stalls at a DNA lesion, the PCNA monoubiquitination by Rad6-Rad18 enables polymerase switch and lesion bypass by the TLS polymerases to occur. Alternatively, polyubiquitination of PCNA by Mms2-Ubc13-Rad5 promotes PRR. In this model, when replication on the leading strand is blocked by a DNA lesion, lagging strand synthesis continues on. Rad5 then gains access to the asymmetric forks and unwinds both the lagging and leading nascent DNA strands, followed by annealing them as well as annealing the template strands. By this fork regression activity a four-way junction intermediate called “chicken foot” is formed. Following that, a DNA polymerase extends the 3′ OH end of the leading nascent strand by copying from the nascent lagging strand. Finally, the back-migration of the four way junction completes error-free replication through the DNA damage.