Reconstitution of recombination-associated DNA synthesis with human proteins - PubMed (original) (raw)
Reconstitution of recombination-associated DNA synthesis with human proteins
Jessica L Sneeden et al. Nucleic Acids Res. 2013 May.
Abstract
The repair of DNA breaks by homologous recombination is a high-fidelity process, necessary for the maintenance of genome integrity. Thus, DNA synthesis associated with recombinational repair must be largely error-free. In this report, we show that human DNA polymerase delta (δ) is capable of robust DNA synthesis at RAD51-mediated recombination intermediates dependent on the processivity clamp PCNA. Translesion synthesis polymerase eta (η) also extends these substrates, albeit far less processively. The single-stranded DNA binding protein RPA facilitates recombination-mediated DNA synthesis by increasing the efficiency of primer utilization, preventing polymerase stalling at specific sequence contexts, and overcoming polymerase stalling caused by topological constraint allowing the transition to a migrating D-loop. Our results support a model whereby the high-fidelity replicative DNA polymerase δ performs recombination-associated DNA synthesis, with translesion synthesis polymerases providing a supportive role as in normal replication.
Figures
Figure 1.
Pathways of homologous recombination involve different modes of DNA synthesis. Homologous recombination (HR) initiates with the common steps of DSB processing, Rad51 nucleofilament assembly, homology search and DNA strand invasion. Following D-loop formation, three different HR pathways are recognized: double Holliday Junction (dHJ), Synthesis-Dependent Strand Annealing (SDSA) and BIR. The initial DNA synthesis is displacement synthesis primed from the 3′-OH end of the invading strand in the D-loop (first end DNA synthesis). There is evidence that this intermediate is already pathway-specific, but it is unclear, how this specification is achieved. In the dHJ pathway, the second end of the DSB is captured by the displaced strand of the D-loop. This second end DNA synthesis does not involve displacement synthesis per se, but after filling the gap may involve displacement synthesis once the extension reaches the 5′-resected end. Likewise in SDSA, after D-loop dissolution and annealing of the extended first strand, the second end synthesis is by a non-displacement mode at least until the gap is filled. In BIR, the requirements are more akin to elongation during replicative DNA synthesis.
Figure 2.
D-loop formation and DNA synthesis. (A) Experimental scheme. The single-stranded 93mer is 32P-endlabeled (asterisk). See also Figure 4A legend for aspects of topology. (B) Analysis of D-loop formation and DNA synthesis by Pol δ and Pol η at D-loops as measured by 0.8 % native agarose gel. Percent D-loops were determined at 0 min. (C) Cartoon depicting analysis of products by two-dimensional gel electrophoresis. (D) Two-dimensional gel electrophoresis of D-loop reactions extended by Pol δ in the presence or absence of RFC, PCNA after 30 min extension time. (E) Same as in D, using Pol η.
Figure 3.
Efficient DNA synthesis by Pol δ. (A) Analysis of DNA synthesis products as measured by 1.2 % alkaline agarose gel electrophoresis with samples after 5 min extension time by polymerases. (B) Quantitation of DNA synthesis products shown in (A), as a function of size of products as well as percent of D-loops extended. Numerical data and errors are in
Supplementary Table S1
. knt: 1000 nucleotides.
Figure 4.
DNA synthesis proceeds via a migrating D-loop. (A) Experimental scheme. Single-stranded 93mer is 32P-endlabeled (asterisk). The dsDNA substrate contains ∼15 negative supercoils (−sc). On D-loop formation with the 93mer, this changes to ∼6 negative supercoils. For each 10.5 nt synthesized, one positive supercoil (+sc) is added, resulting in the accumulation of positive supercoils during D-loop extension. (B) Analysis of D-loop formation and DNA synthesis in the presence or absence of TopoI as measured by 0.8 % native agarose gel electrophoresis. (C) Cartoon depicting analysis of products by two-dimensional gel electrophoresis. (D) Two-dimensional gel electrophoresis of D-loops extended by Pol δ in the presence or absence of TopoI.
Figure 5.
RPA facilitates efficient D-loop extension by Pol δ. (A) Reactions were carried out as described, with RPA titrated at the following concentrations: 0.1, 0.25, 0.5, 1.0, 2.0, 4.0, 6.0 μM. Samples (30 min time point) were analyzed by 1.2 % alkaline agarose gel electrophoresis. (B) Quantitation of synthesis products in (A). Numerical data and errors are in
Supplementary Table S2
. (C) Quantitation of representative sites of replication stalling (0–2 µM: average ± SEM, n = 3). (D) Reactions in (A), analyzed by 5.5 % denaturing acrylamide gel electrophoresis. Arrows indicate examples of G-tracts on the newly synthesized strand that elicit polymerase stalling that is suppressed by RPA. The initial stall region due to topological constraints is suppressed by addition of topoisomerase I (see Figure 4D) and marked on the right hand side.
Figure 6.
Recombination-associated DNA synthesis. RAD51-mediated homology search and DNA strand invasion generates the D-loop intermediate and the invading 3′-OH end serves to initiate recombination-associated DNA synthesis. After dissociation of RAD51 from the heteroduplex DNA, RFC loads PCNA at the 3′-junction enabling binding of DNA polymerase. This process is aided by RPA binding to ssDNA. DNA polymerase initiates DNA synthesis and comes to an initial stall owing to the accumulation of positive supercoils. Extrusion of the 5′-end of the invading DNA alleviates the topological constraint and allows topologically unhindered extension via a migrating D-loop. RPA binding to the template strand in front of DNA polymerase to facilitate processive DNA synthesis.
References
- Lehmann AR, Niimi A, Ogi T, Brown S, Sabbioneda S, Wing JF, Kannouche PL, Green CM. Translesion synthesis: Y-family polymerases and the polymerase switch. DNA Repair. 2007;6:891–899. -PubMed
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