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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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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.
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gkt192-F6: 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.

Mentions: Here, we report the reconstitution of human recombination reactions involving DNA Pol δ, PCNA and RFC. This reconstituted system provides the first evidence of an involvement of human Pol δ in HR and allowed us to generate significant information about novel roles of RPA during HR. First, human Pol δ efficiently extends RAD51-mediated D-loops (Figure 6). Extension of the invading strand is dependent on the processivity clamp PCNA and displacement synthesis can extend over significant distances of several kb. The efficiency of D-loop extension by DNA Pol δ is surprisingly high and indistinguishable from that with canonical primer:templates. These findings are consistent with other in vitro data using human and yeast Pol δ (9,10,14) (L. Krejci, personal communication). Together with compelling genetic evidence in yeast (4–8), these data suggest that DNA Pol δ might be the primary polymerase that extends the invading strand present in a RAD51-mediated D-loop. Based on the reported biochemical properties for calf thymus DNA Pol ε (25), it appears unlikely that Pol ε is involved in first end synthesis during HR, due to its inability to perform strand displacement synthesis. The genetic requirement of HR for DNA Pol ε in yeast (4) may suggest that this DNA polymerase plays a role in second end synthesis during HR but there is currently no experimental evidence for this (Figure 1).Figure 6.


Reconstitution of recombination-associated DNA synthesis with human proteins.

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

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

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

gkt192-F6: 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.
Mentions: Here, we report the reconstitution of human recombination reactions involving DNA Pol δ, PCNA and RFC. This reconstituted system provides the first evidence of an involvement of human Pol δ in HR and allowed us to generate significant information about novel roles of RPA during HR. First, human Pol δ efficiently extends RAD51-mediated D-loops (Figure 6). Extension of the invading strand is dependent on the processivity clamp PCNA and displacement synthesis can extend over significant distances of several kb. The efficiency of D-loop extension by DNA Pol δ is surprisingly high and indistinguishable from that with canonical primer:templates. These findings are consistent with other in vitro data using human and yeast Pol δ (9,10,14) (L. Krejci, personal communication). Together with compelling genetic evidence in yeast (4–8), these data suggest that DNA Pol δ might be the primary polymerase that extends the invading strand present in a RAD51-mediated D-loop. Based on the reported biochemical properties for calf thymus DNA Pol ε (25), it appears unlikely that Pol ε is involved in first end synthesis during HR, due to its inability to perform strand displacement synthesis. The genetic requirement of HR for DNA Pol ε in yeast (4) may suggest that this DNA polymerase plays a role in second end synthesis during HR but there is currently no experimental evidence for this (Figure 1).Figure 6.

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