Browsing by Author "Ira, Grzegorz"

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    Break-induced replication: functions and molecular mechanism
    (Elsevier, 2013) Malkova, Anna; Ira, Grzegorz; Biology, School of Science
    Break-induced replication (BIR) is the pathway of homologous recombination (HR) conserved from phages to eukaryotes that serves to repair DNA breaks that have only one end. BIR contributes to the repair of broken replication forks and allows telomere lengthening in the absence of telomerase. Nonallelic BIR may lead to translocations and other chromosomal rearrangements. In addition, BIR initiated at sites of microhomology can generate copy number variations (CNVs) and complex chromosomal changes. The level of mutagenesis associated with DNA synthesis in BIR is significantly higher than during normal replication. These features make BIR a likely pathway to promote bursts of genetic changes that fuel cancer progression and evolution.
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    Defective Resection at DNA Double-Strand Breaks Leads to De Novo Telomere Formation and Enhances Gene Targeting
    (Public Library of Science, 2010-05-13) Chung, Woo-Hyun; Zhu, Zhu; Papusha, Alma; Malkova, Anna; Ira, Grzegorz; Biology, School of Science
    The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.
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    Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration
    (Springer Nature, 2013) Wilson, Marenda A.; Kwon, YoungHo; Xu, Yuanyuan; Chung, Woo-Hyun; Chi, Peter; Niu, Hengyao; Mayle, Ryan; Chen, Xuefeng; Malkova, Anna; Sung, Patrick; Ira, Grzegorz; Biology, School of Science
    During DNA repair by homologous recombination (HR), DNA synthesis copies information from a template DNA molecule. Multiple DNA polymerases have been implicated in repair-specific DNA synthesis, but it has remained unclear whether a DNA helicase is involved in this reaction. A good candidate DNA helicase is Pif1, an evolutionarily conserved helicase in Saccharomyces cerevisiae important for break-induced replication (BIR) as well as HR-dependent telomere maintenance in the absence of telomerase found in 10-15% of all cancers. Pif1 has a role in DNA synthesis across hard-to-replicate sites and in lagging-strand synthesis with polymerase δ (Polδ). Here we provide evidence that Pif1 stimulates DNA synthesis during BIR and crossover recombination. The initial steps of BIR occur normally in Pif1-deficient cells, but Polδ recruitment and DNA synthesis are decreased, resulting in premature resolution of DNA intermediates into half-crossovers. Purified Pif1 protein strongly stimulates Polδ-mediated DNA synthesis from a D-loop made by the Rad51 recombinase. Notably, Pif1 liberates the newly synthesized strand to prevent the accumulation of topological constraint and to facilitate extensive DNA synthesis via the establishment of a migrating D-loop structure. Our results uncover a novel function of Pif1 and provide insights into the mechanism of HR.