Substrate-selective repair and restart of replication forks by DNA translocases

Abstract

Stalled replication forks are sources of genetic instability. Multiple fork-remodeling enzymes are recruited to stalled forks, but how they work to promote fork restart is poorly understood. By combining ensemble biochemical assays and single-molecule studies with magnetic tweezers, we show that SMARCAL1 branch migration and DNA-annealing activities are directed by the single-stranded DNA-binding protein RPA to selectively regress stalled replication forks caused by blockage to the leading-strand polymerase and to restore normal replication forks with a lagging-strand gap. We unveil the molecular mechanisms by which RPA enforces SMARCAL1 substrate preference. E. coli RecG acts similarly to SMARCAL1 in the presence of E. coli SSB, whereas the highly related human protein ZRANB3 has different substrate preferences. Our findings identify the important substrates of SMARCAL1 in fork repair, suggest that RecG and SMARCAL1 are functional orthologs, and provide a comprehensive model of fork repair by these DNA translocases.

Figure 1

RPA directs SMARCAL1 specifically to a damaged replication fork substrate and inhibits its action at a normal fork. (A,C) Diagram of the lagging (A) or leading (C) gap replication fork regression assay. ³²P-labeled strands are indicated with asterisks. A two base-pair mismatch is present on the parental (black) strands to prevent spontaneous branch migration. The physiological reaction mimicked by the experimental assay is shown in brackets. (B,D) Lagging (B) or leading (D) gap replication fork substrates were incubated 15min at room temperature in the presence or absence of RPA. Increasing amount of SMARCAL1 WT or Δ34 were added to the reaction and further incubated 20min at 30°C. DNA products were analyzed by native gel electrophoresis and phosphorimager quantitation of three experiments is shown (mean +/− SD). See also Fig. S1.

Figure 2

SMARCAL1 binds asymmetrically to double-stranded DNA on the gapped fork substrates. (A,B) In each experiment, increasing amounts of SMARCAL1 WT or Δ34 was incubated with the labeled substrate for 15min prior to addition of nuclease. A diagram of each DNA substrate is placed next to the gel and stretched to correspond to the location of the size standards. The ³²P-labelled DNA strand is indicated with an asterisk (*). Where indicated RPA was pre-bound to the substrate at a concentration sufficient to yield 100% binding. After nuclease digestion at levels titrated to yield single cleavage events per DNA substrate, the reaction products were heat denatured and separated on an 8% polyacrylamide denaturing sequencing gel. The control (Ctl) samples are the DNA substrates in the absence of SMARCAL1 protein. The stars and boxes next to the DNA substrate indicate nuclease hypersensitive and protected regions induced by SMARCAL1. (C) Summary of the modification of the digestion patterns observed in footprinting studies.

Figure 3

SMARCAL1 catalyzes repetitive rounds of DNA annealing. (A) Schematic of the magnetic tweezers single molecule annealing experiment. Details are described in supplemental methods. (B and C) Experimental traces corresponding to background fluctuation (green) or SMARCAL1 annealing activity (blue). Example of repetitive annealing events catalyzed by single SMARCAL1 molecules at 30pM enzyme concentration is shown. Panel C is a zoom in of a small portion of panel B. (D, E) Mean annealing rates (D) and processivity (E) of SMARCAL1 wild type (WT) or RPA-binding mutant (Δ34) in presence or absence of RPA calculated by fitting the distribution of rates and processivity to a Gaussian function and exponential function respectively. At least 75 traces were analyzed per condition. See also Fig. S3.

Figure 4

RPA increases the distance SMARCAL1 travels per annealing event specifically on a DNA substrate that mimics a stalled replication fork with a leading strand gap. (A) Schematics of the strand displacement substrate construction with a gap on the lagging or leading strand. Details are described in Extended Experimental Procedures and Fig. S4. (B) Example of a typical experimental trace displaying characteristic features of the strand displacement reaction (SMARCAL1 binding, SMARCAL1 repetitive annealing, full oligonucleotide displacement and final hairpin annealing) at 200pM enzyme concentration. The molecular extensions corresponding to the initial strand displacement substrate and the fully formed hairpin are highlighted in blue and green respectively. (C) The ratio of T-rep measured in the absence or presence of RPA was measured at three different F_test forces for both lagging and leading strand gap substrates. (n = 58–89 depending on condition). Error bars represent SD. The experiment at 10pN was done with a wash step after RPA binding to remove any free RPA molecules excluding the possibility that free RPA contributes to the differences. (D) Typical trace of the leading strand gap substrate showing repetitive annealing events in absence or presence of RPA. ΔZ represent the amplitude of the repetitive annealing events measured in number of annealed base pairs. (E) Mean and SD of ΔZ in the absence or presence of RPA for lagging or leading strand gap substrates. The difference in ΔZ measured in assays in absence and presence of RPA is significant (independent 2-group t-test: P=0.006 and P= 1.9e–07 for lagging and leading strand gap substrates). (F) The presence of a second oligonucleotide to generate a true 64 bp gap on the lagging or leading strand did not change the ability of RPA to inhibit or activate SMARCAL1 respectively. See also Fig. S4.

Figure 5

SMARCAL1 preferentially catalyzes fork restoration that yields a normal lagging strand gap replication fork in the presence of RPA. (A,C) Schematic of the leading (A) or lagging (C) gap replication fork restoration assay. ³²P-labeled strands are indicated with asterisks. Mismatches in the DNA strands were inserted between the longest nascent strand and the corresponding parental strand to prevent spontaneous fork restoration. In brackets is the physiological reaction mimicked by the experimental assay. (B,D) Leading (B) or lagging (D) gap replication fork restoration substrates were incubated 15min at room temperature in the presence or absence of RPA sufficient to bind 100% of the DNA substrate. Increasing amounts of SMARCAL1 WT or Δ34 were added to the reaction and further incubated 20min. DNA products were analyzed by native gel electrophoresis. Mean +/− SD from three independent experiments is depicted. See also Fig. S6 and Extended Experimental Results.

Figure 6

E. coli RecG but not human ZRANB3 exhibits similar substrate preferences to SMARCAL1 in the presence of ssDNA binding proteins. (A,C) Leading or lagging strand gap fork regression substrates were incubated 15min in the presence or absence of RPA (A) or SSB (C). Increasing amounts of ZRANB3 (A) or RecG (C) were then added to the reaction and further incubated 20min. The DNA products were analyzed by native gel electrophoresis. (B,D) Fork restoration substrates were incubated with RPA or SSB and ZRANB3 or RecG as indicated. Reaction products were analyzed by native gel electrophoresis. Mean +/−SD from three experiments is shown in all graphs.

Figure 7

Model for damaged replication fork repair by SMARCAL1. (i–ii) RPA inhibits SMARCAL1 from regressing normal elongating forks with lagging-strand template gaps. DNA damage on the leading-strand template induces stalling of the replicative DNA polymerase and generation of a leading-strand ssDNA. (ii–iii) RPA stimulates SMARCAL1 fork regression activity on this stalled fork. (iii–iv) Continued branch migration yields a true chicken foot structure. (iv–v) Fork regression permits repair of the DNA lesion in the context of dsDNA or could allow template switching. The nascent lagging-strand of the regressed fork can be digested by a 5′ to 3 ′ exonuclease, which may form regressed fork with a longer nascent leading strand. (v–vi) Strand switching and branch migration would yield a partially regressed replication fork with a ssDNA strand 3 ′ tail corresponding to the nascent leading-strand. (vi–i) RPA stimulates SMARCAL1 to re-anneal the nascent leading ssDNA strand with the complementary parental strand to reform a normal DNA replication fork that can resume DNA replication. This model is similar to those described for E. coli RecG function (Gregg et al., 2002; McGlynn and Lloyd, 2000), although it also incorporates the substrate specificity dictated by single-stranded DNA binding proteins.