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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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Related in: MedlinePlus

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.
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gkt192-F1: 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.

Mentions: Homology-directed repair of double-stranded DNA breaks (DSB) is essential to maintain genomic integrity. Homologous recombination (HR) is a high-fidelity repair process, and deficiencies in genes involved in recombination have been shown to predispose individuals to tumor formation (1). In HR-mediated repair, a DSB is processed to generate 3′-terminal single-stranded DNA (ssDNA), which is then bound by the ssDNA binding protein RPA (see Figure 1). Formation of the RAD51 nucleoprotein filament on such substrates involves RAD51 paralogs, BRCA2 and other factors, whereupon homology search takes place. The 3′-ssDNA invades the homologous donor double-stranded DNA (dsDNA) to initiate DNA strand exchange. At this stage, DNA synthesis primed by the invading strand occurs (first end synthesis), after which the recombination intermediate can be processed in a number of ways, leading to non-crossover or crossover outcomes. The precise mechanisms by which DNA synthesis takes place during HR are not fully understood. In humans, 15 different DNA polymerases have been identified (2). The primary replicative polymerases epsilon (ε) and delta (δ) are responsible for the bulk of genomic nuclear replication, performing synthesis on the leading and lagging strands, respectively. Pol ε and Pol δ are the most high-fidelity polymerases in mammals, generating less than one error per 105 base pairs synthesized; they also possess 3′–5′ proofreading exonuclease activities capable of excising misincorporated bases, rendering replicative synthesis a highly faithful process (2). On encountering a synthesis-blocking lesion, replicative polymerases dissociate and translesion synthesis (TLS) polymerases can be recruited to bypass these lesions (3). Unlike replicative polymerases, TLS polymerases lack proofreading exonuclease activity and synthesize DNA in both a distributive and low-fidelity manner (2). These properties of TLS polymerases allow them to bypass bulky or uninformative lesions in DNA, sacrificing fidelity to prevent DNA breaks that may lead to chromosomal rearrangements and tumorigenesis (2).Figure 1.


Reconstitution of recombination-associated DNA synthesis with human proteins.

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

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

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

gkt192-F1: 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.
Mentions: Homology-directed repair of double-stranded DNA breaks (DSB) is essential to maintain genomic integrity. Homologous recombination (HR) is a high-fidelity repair process, and deficiencies in genes involved in recombination have been shown to predispose individuals to tumor formation (1). In HR-mediated repair, a DSB is processed to generate 3′-terminal single-stranded DNA (ssDNA), which is then bound by the ssDNA binding protein RPA (see Figure 1). Formation of the RAD51 nucleoprotein filament on such substrates involves RAD51 paralogs, BRCA2 and other factors, whereupon homology search takes place. The 3′-ssDNA invades the homologous donor double-stranded DNA (dsDNA) to initiate DNA strand exchange. At this stage, DNA synthesis primed by the invading strand occurs (first end synthesis), after which the recombination intermediate can be processed in a number of ways, leading to non-crossover or crossover outcomes. The precise mechanisms by which DNA synthesis takes place during HR are not fully understood. In humans, 15 different DNA polymerases have been identified (2). The primary replicative polymerases epsilon (ε) and delta (δ) are responsible for the bulk of genomic nuclear replication, performing synthesis on the leading and lagging strands, respectively. Pol ε and Pol δ are the most high-fidelity polymerases in mammals, generating less than one error per 105 base pairs synthesized; they also possess 3′–5′ proofreading exonuclease activities capable of excising misincorporated bases, rendering replicative synthesis a highly faithful process (2). On encountering a synthesis-blocking lesion, replicative polymerases dissociate and translesion synthesis (TLS) polymerases can be recruited to bypass these lesions (3). Unlike replicative polymerases, TLS polymerases lack proofreading exonuclease activity and synthesize DNA in both a distributive and low-fidelity manner (2). These properties of TLS polymerases allow them to bypass bulky or uninformative lesions in DNA, sacrificing fidelity to prevent DNA breaks that may lead to chromosomal rearrangements and tumorigenesis (2).Figure 1.

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