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Reconstitution of recombination-associated DNA synthesis with human proteins.

Sneeden JL, Grossi SM, Tappin I, Hurwitz J, Heyer WD - Nucleic Acids Res. (2013)

Bottom Line: 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.Translesion synthesis polymerase eta (η) also extends these substrates, albeit far less processively.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, CA 95616-8665, USA.

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.

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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.
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gkt192-F4: 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.

Mentions: 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.


Reconstitution of recombination-associated DNA synthesis with human proteins.

Sneeden JL, Grossi SM, Tappin I, Hurwitz J, Heyer WD - Nucleic Acids Res. (2013)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3643601&req=5

gkt192-F4: 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.
Mentions: 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.

Bottom Line: 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.Translesion synthesis polymerase eta (η) also extends these substrates, albeit far less processively.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology & Molecular Genetics, University of California, Davis, Davis, CA 95616-8665, USA.

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.

Show MeSH
Related in: MedlinePlus