Pif1 helicase and Polδ promote recombination-coupled DNA synthesis via bubble migration

• Published in: Nature (London) 2013-10, Vol.502 (7471), p.393-396
• Main Authors: Wilson, Marenda A., Kwon, YoungHo, Xu, Yuanyuan, Chung, Woo-Hyun, Chi, Peter, Niu, Hengyao, Mayle, Ryan, Chen, Xuefeng, Malkova, Anna, Sung, Patrick, Ira, Grzegorz
• Format: Article
• Language: English
• Subjects: 631/208/211; 631/337/149; 631/337/151/1431; 631/45/147; Crossing Over, Genetic; DNA; DNA Helicases - deficiency; DNA Helicases - genetics; DNA Helicases - metabolism; DNA Polymerase III - metabolism; DNA Repair; DNA Replication; DNA synthesis; DNA, Fungal - biosynthesis; DNA, Fungal - chemistry; DNA, Fungal - metabolism; Genetic recombination; Genetic research; Helicases; Humanities and Social Sciences; letter; multidisciplinary; Nucleic Acid Conformation; Physiological aspects; Rad51 Recombinase - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Science; Telomerase
• ISSN: 0028-0836; 1476-4687
• DOI: 10.1038/nature12585

This paper demonstrates that Pif1 helicase works with polymerase d to promote DNA synthesis through a migrating D-loop, a mechanism used to copy tens of kilobases during repair of chromosome breaks by break-induced replication (BIR). Pif1 helicase promotes BIR-specific DNA synthesis When DNA is repaired by homologous recombination, DNA synthesis is involved in the latter stages. Two papers published in this issue of Nature now define a role for the DNA helicase Pif1 in this reaction. They show that although the initial stages of break-induced replication (BIR) can occur normally in the absence of Pif1, synthesis from a migrating D-loop intermediate is compromised. The mechanism of replication during BIR involves a unique bubble-like replication fork that results in conservative inheritance of the new genetic material, in contrast to the S-phase replication that duplicates the genome before cell division, and is inherently mutagenic. 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 1 , 2 , 3 , 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) 4 as well as HR-dependent telomere maintenance in the absence of telomerase 5 found in 10–15% of all cancers 6 . Pif1 has a role in DNA synthesis across hard-to-replicate sites 7 , 8 and in lagging-strand synthesis with polymerase δ (Polδ) 9 , 10 , 11 . 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.