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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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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.
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gkt192-F2: 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.

Mentions: As human Pol δ requires the accessory factor PCNA and its clamp loader RFC for efficient synthesis of canonical primer-template substrates, we rationalized that these factors would also be necessary for efficient synthesis during recombination-associated DNA synthesis (23,24). To investigate the role of replication factors in DNA synthesis of recombination intermediates, we reconstituted an in vitro DSB repair system using human proteins (RAD51, RPA, Pol δ, PCNA, and RFC) in a plasmid-based D-loop assay (Figure 2A). The reaction was carried out by first incubating RAD51 and RPA with a 5′-endlabeled 93mer ssDNA in the presence of calcium ions to form competent nucleoprotein filaments (37). Negatively supercoiled plasmid (pUC19, 2686 bp) was added to initiate D-loop formation. Calcium is required for efficient filament formation by hRAD51, and chelation is necessary to allow ATP hydrolysis, necessary for maximum D-loop formation, and to allow for DNA polymerases to be active. RFC, PCNA and polymerase were added to initiate DNA synthesis. As previously demonstrated, in vitro D-loop formation and DNA synthesis using human proteins does not require RAD54 (38).Figure 2.


Reconstitution of recombination-associated DNA synthesis with human proteins.

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

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

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

gkt192-F2: 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.
Mentions: As human Pol δ requires the accessory factor PCNA and its clamp loader RFC for efficient synthesis of canonical primer-template substrates, we rationalized that these factors would also be necessary for efficient synthesis during recombination-associated DNA synthesis (23,24). To investigate the role of replication factors in DNA synthesis of recombination intermediates, we reconstituted an in vitro DSB repair system using human proteins (RAD51, RPA, Pol δ, PCNA, and RFC) in a plasmid-based D-loop assay (Figure 2A). The reaction was carried out by first incubating RAD51 and RPA with a 5′-endlabeled 93mer ssDNA in the presence of calcium ions to form competent nucleoprotein filaments (37). Negatively supercoiled plasmid (pUC19, 2686 bp) was added to initiate D-loop formation. Calcium is required for efficient filament formation by hRAD51, and chelation is necessary to allow ATP hydrolysis, necessary for maximum D-loop formation, and to allow for DNA polymerases to be active. RFC, PCNA and polymerase were added to initiate DNA synthesis. As previously demonstrated, in vitro D-loop formation and DNA synthesis using human proteins does not require RAD54 (38).Figure 2.

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