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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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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.
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gkt192-F3: 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.

Mentions: The reconstituted human D-loop formation/extension reactions were efficient, generating ∼30% D-loops (Figure 2B). Pol δ is capable of robust D-loop extension, and recombination-associated DNA synthesis is dependent on the presence of RAD51, PCNA and RFC (Figure 2B and D). In the presence of these factors and RPA, after 5 min incubation, Pol δ extended on average 48% of total primer termini engaged in D-loops, with 14% of products >400 nt in length, and 2% of products over 2 kb (Figure 3A and B, lane 6). We routinely observed products as long as 10 000 nt in these assays after 5 min incubation; longer products were predominant after 30 min incubation (Figure 2D and Supplementary Figure S1). This is significantly longer than the 2686 bp duplex DNA template and demonstrates that DNA synthesis in these reactions is processive and proceeds as a rolling circle.Figure 3.


Reconstitution of recombination-associated DNA synthesis with human proteins.

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

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

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

gkt192-F3: 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.
Mentions: The reconstituted human D-loop formation/extension reactions were efficient, generating ∼30% D-loops (Figure 2B). Pol δ is capable of robust D-loop extension, and recombination-associated DNA synthesis is dependent on the presence of RAD51, PCNA and RFC (Figure 2B and D). In the presence of these factors and RPA, after 5 min incubation, Pol δ extended on average 48% of total primer termini engaged in D-loops, with 14% of products >400 nt in length, and 2% of products over 2 kb (Figure 3A and B, lane 6). We routinely observed products as long as 10 000 nt in these assays after 5 min incubation; longer products were predominant after 30 min incubation (Figure 2D and Supplementary Figure S1). This is significantly longer than the 2686 bp duplex DNA template and demonstrates that DNA synthesis in these reactions is processive and proceeds as a rolling circle.Figure 3.

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