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. 2016 Jul 8;44(12):5758-72.
doi: 10.1093/nar/gkw339. Epub 2016 Apr 29.

Dynamic binding of replication protein a is required for DNA repair

Ran Chen  1 , Shyamal Subramanyam  2 , Adrian H Elcock  1 , Maria Spies  1 , Marc S Wold  3
Affiliations

Dynamic binding of replication protein a is required for DNA repair

Ran Chen et al. Nucleic Acids Res. .

Abstract

Replication protein A (RPA), the major eukaryotic single-stranded DNA (ssDNA) binding protein, is essential for replication, repair and recombination. High-affinity ssDNA-binding by RPA depends on two DNA binding domains in the large subunit of RPA. Mutation of the evolutionarily conserved aromatic residues in these two domains results in a separation-of-function phenotype: aromatic residue mutants support DNA replication but are defective in DNA repair. We used biochemical and single-molecule analyses, and Brownian Dynamics simulations to determine the molecular basis of this phenotype. Our studies demonstrated that RPA binds to ssDNA in at least two modes characterized by different dissociation kinetics. We also showed that the aromatic residues contribute to the formation of the longer-lived state, are required for stable binding to short ssDNA regions and are needed for RPA melting of partially duplex DNA structures. We conclude that stable binding and/or the melting of secondary DNA structures by RPA is required for DNA repair, including RAD51 mediated DNA strand exchange, but is dispensable for DNA replication. It is likely that the binding modes are in equilibrium and reflect dynamics in the RPA-DNA complex. This suggests that dynamic binding of RPA to DNA is necessary for different cellular functions.

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Figures

Figure 1.
Figure 1.
Surface-tethered RPA DNA-binding activity. (A) Schematic representation of TIRFM-based assay for analysis of DNA binding by tethered RPA. RPA is immobilized on the surface of the microscope flow cell and an evanescent field generated on cell surface by TIR while illuminating with a 530 nM laser. The DNA substrates labeled with Cy3 are only visible when bound to surface-tethered RPA inside the evanescent field. (B) A representative trajectory one RPA molecule monitored in the presence of Cy3-labeled dT35 for 6000 s. Each dot represents a fluorescence reading and black line represents the two-state model determined by QUB. Spikes in the fluorescence intensity correspond to binding and dissociation of different DNA molecules. The length of each binding event is an ‘on time’, and time between binding events is counted as an ‘off time’. In each experiment, 600–1000 trajectories from individual surface-tethered RPA molecules similar to the one depicted here are analyzed. (C) kon and (D) koff determined for RPA binding to dT35 using smTIRF. Distributions of ‘on times’ and ‘off times’ were fit to single or double exponential decay equations to obtain von decay and koff-fast, koff-slow, and koff as shown. The von decay was determined at concentration 0.1, 0.2 and 0.3 nM. The plot shows that von decay had a linear relationship with DNA concentration; kon (s−1M−1) is the slope.
Figure 2.
Figure 2.
Length and structure dependence of DNA binding. (A) Binding affinity of RPA and Aro mutants to the indicated DNA substrates was determined by smTIRF. Association constants from two-phase exponential decay, Kafast (A) and Kaslow (B) were calculated based on kon and koff-fast and koff-slow from (Supplementary Table S1B). Data from AroA, AroB and Aro2 binding with dT20 fit best to a one phase exponential decay. So the association constant (Kaone phase) calculated from the kon and koff (Supplementary Table S1A) is shown in (C). No binding events were detected (NE) for AroA, AroB and Aro2 with dT15. Error bars represent standard deviation of three experiments.
Figure 3.
Figure 3.
Fraction of long-lived RPA–DNA complexes. (A) The ‘dwell time’ distribution for RPA binding to dT35, dT25, dT20 and dT15 with a 40-s cutoff indicated (dashed line). (B) Quantification of the fraction of events with dwell-times longer than 40 s for RPA, AroA, AroB and Aro2 with each of the oligonucleotides analyzed. Error bars indicate standard errors from three experiments. No long-dwell complex (>40 s) were detected AroB and Aro2 with dT20 (ND) and no binding events were observed with the Aro mutants with dT15 (NE).
Figure 4.
Figure 4.
DNA-binding to and helix destablization of a replication fork like (RFL) DNA. (A) RFL-binding and (B) helix destabilization activities of RPA and Aro mutants. The indicated amount of RPA or Aro mutant was incubated with 6 nM of RFL at 25° for 25 min. The reactions were separated by electrophoresis under binding (A) or helix destabilization (B) conditions. The positions of the DNA–protein complex, free (RFL) DNA, RFL and ssDNA are indicated. Slow mobility complex indicated with *. The boiled control (B) confirms the position of melted Cy3- and Cy5-labeled ssDNAs. Schematic shows positions of Cy3 (light) and Cy5 (dark) in RFL with arrowheads indicating 3′ ends.
Figure 5.
Figure 5.
DNA-binding to and helix destablization of a 20 nt Bubble DNA. (A) Bubble binding and (B) helix destabilization activities of RPA and Aro mutants. The indicated amount of RPA or Aro mutant was incubated with 6 nM of fluorophore-labeled bubble at 25° for 25 min. The reactions were terminated and separated by electrophoresis under binding (A) or helix destabilization (B) conditions. The positions of the DNA-protein complex, free (Bubble) DNA, Bubble DNA and ssDNA are indicated. The boiled control (B) confirms the position of the position of the Cy3-labeled ssDNA. Schematic shows position of Cy3 in Bubble DNA with arrowheads indicating 3′ ends.
Figure 6.
Figure 6.
Brownian dynamic modeling of RPA interactions with Gap and Bubble DNA. (A) Representative simulation of RPA with Gap or Bubble DNA. Top-schematic of initial conformation, middle-initial conformation and bottom-final conformation after 5 μs. RPA subunits (RPA1, RPA2 and RPA3) shown in green, blue and red, respectively; DNA shown in gray (bases and deoxyribose) and pink (phosphates). *Melted, non-RPA bound strand of DNA. (B) Average change in percent contacts (Q-value) for DNA-DNA (green) or RPA-DNA (red) over five independent 5 μs simulations for Gap and Bubble DNAs. Error bars represent standard deviation.
Figure 7.
Figure 7.
Function of Aro mutants in Rad51 mediated strand exchange. (A) Schematic of the RAD51 DNA strand exchange assay. RAD51 coated φX174 circular ssDNA recombines with φX174 RFI linear dsDNA. This leads to formation of joint molecules followed by the nicked circular product, containing the ssDNA circular substrate and the complementary ssDNA strand, and the displaced ssDNA strand. (B) Reactions containing indicated amounts of wild-type or Aro mutant forms of RPA were assembled (0 min) and incubated at 37° for 60 min. Positions of starting DNA (linear duplex), initially formed joint molecule intermediates and completely exchanged nicked circular product are indicated. Some denatured ssDNA (bottom of gel) was present in the starting DNA (0-min points).
Figure 8.
Figure 8.
Model of possible dynamic RPA binding modes. See Discussion for details.
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