DNA2 drives processing and restart of reversed replication forks in human cells

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Abstract

Following prolonged genotoxic stress, DNA2 and WRN functionally interact to degrade reversed replication forks and promote replication restart, thereby preventing aberrant processing of unresolved replication intermediates

Abstract

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5′-to-3′ polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.

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Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.

Jae-Woo Chung, Zhu Zhu, Sang Lee … (2008)

Formation of single-strand DNA (ssDNA) tails at a double-strand break (DSB) is a key step in homologous recombination and DNA-damage signaling. The enzyme(s) producing ssDNA at DSBs in eukaryotes remain unknown. We monitored 5'-strand resection at inducible DSB ends in yeast and identified proteins required for two stages of resection: initiation and long-range 5'-strand resection. We show that the Mre11-Rad50-Xrs2 complex (MRX) initiates 5' degradation, whereas Sgs1 and Dna2 degrade 5' strands exposing long 3' strands. Deletion of SGS1 or DNA2 reduces resection and DSB repair by single-strand annealing between distant repeats while the remaining long-range resection activity depends on the exonuclease Exo1. In exo1Deltasgs1Delta double mutants, the MRX complex together with Sae2 nuclease generate, in a stepwise manner, only few hundred nucleotides of ssDNA at the break, resulting in inefficient gene conversion and G2/M damage checkpoint arrest. These results provide important insights into the early steps of DSB repair in eukaryotes.

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Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.

Eleni Mimitou, Lorraine S. Symington (2008)

DNA ends exposed after introduction of double-strand breaks (DSBs) undergo 5'-3' nucleolytic degradation to generate single-stranded DNA, the substrate for binding by the Rad51 protein to initiate homologous recombination. This process is poorly understood in eukaryotes, but several factors have been implicated, including the Mre11 complex (Mre11-Rad50-Xrs2/NBS1), Sae2/CtIP/Ctp1 and Exo1. Here we demonstrate that yeast Exo1 nuclease and Sgs1 helicase function in alternative pathways for DSB processing. Novel, partially resected intermediates accumulate in a double mutant lacking Exo1 and Sgs1, which are poor substrates for homologous recombination. The early processing step that generates partly resected intermediates is dependent on Sae2. When Sae2 is absent, in addition to Exo1 and Sgs1, unprocessed DSBs accumulate and homology-dependent repair fails. These results suggest a two-step mechanism for DSB processing during homologous recombination. First, the Mre11 complex and Sae2 remove a small oligonucleotide(s) from the DNA ends to form an early intermediate. Second, Exo1 and/or Sgs1 rapidly process this intermediate to generate extensive tracts of single-stranded DNA that serve as substrate for Rad51.

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The Bloom's syndrome helicase suppresses crossing over during homologous recombination.

Leonard Wu, Ian D. Hickson (2003)

Mutations in BLM, which encodes a RecQ helicase, give rise to Bloom's syndrome, a disorder associated with cancer predisposition and genomic instability. A defining feature of Bloom's syndrome is an elevated frequency of sister chromatid exchanges. These arise from crossing over of chromatid arms during homologous recombination, a ubiquitous process that exists to repair DNA double-stranded breaks and damaged replication forks. Whereas crossing over is required in meiosis, in mitotic cells it can be associated with detrimental loss of heterozygosity. BLM forms an evolutionarily conserved complex with human topoisomerase IIIalpha (hTOPO IIIalpha), which can break and rejoin DNA to alter its topology. Inactivation of homologues of either protein leads to hyper-recombination in unicellular organisms. Here, we show that BLM and hTOPO IIIalpha together effect the resolution of a recombination intermediate containing a double Holliday junction. The mechanism, which we term double-junction dissolution, is distinct from classical Holliday junction resolution and prevents exchange of flanking sequences. Loss of such an activity explains many of the cellular phenotypes of Bloom's syndrome. These results have wider implications for our understanding of the process of homologous recombination and the mechanisms that exist to prevent tumorigenesis.

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Author and article information

Journal

Journal ID (nlm-ta): J Cell Biol

Journal ID (iso-abbrev): J. Cell Biol

Journal ID (hwp): jcb

Journal ID (publisher-id): jcb

Title: The Journal of Cell Biology

Publisher: The Rockefeller University Press

ISSN (Print): 0021-9525

ISSN (Electronic): 1540-8140

Publication date (Print): 2 March 2015

Volume: 208

Issue: 5

Pages: 545-562

Affiliations

[1 ]Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, St. Louis, MO 63104

[2 ]Institute of Molecular Cancer Research, University of Zurich, CH-8057 Zurich, Switzerland

[3 ]Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, MO 63110

[4 ]Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55455

Author notes

Correspondence to Alessandro Vindigni: avindign@ 123456slu.edu

[*]

S. Thangavel and M. Berti contributed equally to this paper.

Article

Publisher ID: 201406100

DOI: 10.1083/jcb.201406100

PMC ID: 4347643

PubMed ID: 25733713

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This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

DNA2 drives processing and restart of reversed replication forks in human cells

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Most cited references 46

Sgs1 helicase and two nucleases Dna2 and Exo1 resect DNA double-strand break ends.

Sae2, Exo1 and Sgs1 collaborate in DNA double-strand break processing.

The Bloom's syndrome helicase suppresses crossing over during homologous recombination.

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