INO80-dependent chromatin remodeling regulates early and late stages of mitotic homologous recombination

Department of Molecular Genetics and Microbiology, University of New Mexico School of Medicine and Cancer Center, Albuquerque, NM 87131, United States.
DNA Repair (Impact Factor: 3.11). 03/2009; 8(3):360-9. DOI: 10.1016/j.dnarep.2008.11.014
Source: PubMed


Chromatin remodeling is emerging as a critical regulator of DNA repair factor access to DNA damage, and optimum accessibility of these factors is a major determinant of DNA repair outcome. Hence, chromatin remodeling is likely to play a key role in genome stabilization and tumor suppression. We previously showed that nucleosome eviction near double-strand breaks (DSBs) in yeast is regulated by the INO80 nucleosome remodeling complex and is defective in mutants lacking the Arp8 subunit of INO80. In the absence of homologous donor sequences, RPA recruitment to a DSB appeared normal in arp8Delta, but Rad51 recruitment was defective. We now show that the early strand invasion step of homologous recombination (HR) is markedly delayed in an arp8Delta haploid, but there is only a minor defect in haploid HR efficiency (MAT switching). In an arp8Delta diploid, interhomolog DSB repair by HR shows a modest defect that is partially suppressed by overexpression of Rad51 or its mediator, Rad52. In wild type cells, DSB repair typically results in gene conversion, and most gene conversion tracts are continuous, reflecting efficient mismatch repair of heteroduplex DNA. In contrast, arp8Delta gene conversion tracts are longer and frequently discontinuous, indicating defects in late stages of HR. Interestingly, when a homologous donor sequence is present, Rad51 is recruited normally to a DSB in arp8Delta, but its transfer to the donor is delayed, and this correlates with defective displacement of donor nucleosomes. We propose that retained nucleosomes at donors destabilize heteroduplex DNA or impair mismatch recognition, reflected in delayed strand invasion and altered conversion tracts.

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    • "This reduction in HR raised the question of whether EEPD1 depletion increased gene conversion tract lengths. Cells with defects in HR components display longer gene conversion tracts among residual HR products[30,31,40414243444546. Consistent with these prior studies, HR products from EEPD1-depleted cells had significantly longer conversion tracts compared to controls (Fig 3C and 3D). "
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    ABSTRACT: Replication fork stalling and collapse is a major source of genome instability leading to neoplastic transformation or cell death. Such stressed replication forks can be conservatively repaired and restarted using homologous recombination (HR) or non-conservatively repaired using micro-homology mediated end joining (MMEJ). HR repair of stressed forks is initiated by 5' end resection near the fork junction, which permits 3' single strand invasion of a homologous template for fork restart. This 5' end resection also prevents classical non-homologous end-joining (cNHEJ), a competing pathway for DNA double-strand break (DSB) repair. Unopposed NHEJ can cause genome instability during replication stress by abnormally fusing free double strand ends that occur as unstable replication fork repair intermediates. We show here that the previously uncharacterized Exonuclease/Endonuclease/Phosphatase Domain-1 (EEPD1) protein is required for initiating repair and restart of stalled forks. EEPD1 is recruited to stalled forks, enhances 5' DNA end resection, and promotes restart of stalled forks. Interestingly, EEPD1 directs DSB repair away from cNHEJ, and also away from MMEJ, which requires limited end resection for initiation. EEPD1 is also required for proper ATR and CHK1 phosphorylation, and formation of gamma-H2AX, RAD51 and phospho-RPA32 foci. Consistent with a direct role in stalled replication fork cleavage, EEPD1 is a 5' overhang nuclease in an obligate complex with the end resection nuclease Exo1 and BLM. EEPD1 depletion causes nuclear and cytogenetic defects, which are made worse by replication stress. Depleting 53BP1, which slows cNHEJ, fully rescues the nuclear and cytogenetic abnormalities seen with EEPD1 depletion. These data demonstrate that genome stability during replication stress is maintained by EEPD1, which initiates HR and inhibits cNHEJ and MMEJ.
    Full-text · Article · Dec 2015 · PLoS Genetics
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    • "Studies in Arabidopsis thaliana implicate INO80-C in ectopic (transposon) recombination events [100], and arp8-depleted diploid yeast cells were modestly defective in interhomolog DSB repair by HR [101]. Indeed, in arp8Δ cells, Rad51 is recruited to the DSB but the transfer to the homologous donor showed a marked delay, which correlates with a failure to displace nucleosomes at the donor locus [101]. Thus, it was argued that INO80-C contributes both to very early and very late stages of HR. "
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    ABSTRACT: The double membrane of the eukaryotic nucleus surrounds the genome, constraining it to a nuclear sphere. Proteins, RNA protein particles and artificial chromosome rings diffuse rapidly and freely throughout the nucleoplasm, while chromosomal loci show subdiffusive movement with varying degrees of constraint. In situ biochemical approaches and live imaging studies have revealed the existence of nuclear subcompartments that are enriched for specific chromatin states and/or enzymatic activities. This sequestration is thought to enhance the formation of heterochromatin, particularly when factors of limited abundance are involved. Implicit in the concept of compartmentation is the idea that chromatin is able to move from one compartment to another. Indeed, in budding yeast, gene activation, repression and the presence of persistent DNA double-strand breaks each has been shown to provoke subnuclear relocalization of chromatin. In some cases, movement has been linked to the action of ATP-dependent chromatin remodeling complexes, more specifically to the Snf2-related ATPase-containing complexes, SWR-C and INO80-C. Here we examine how these multi-subunit remodelers contribute to chromatin-based processes linked to the DNA damage response. We review recent evidence that supports a role for yeast SWR-C and INO80-C in determining the subnuclear position of damaged domains and finally, we recap the multiple ways in which these remodelers contribute to genomic integrity.
    Full-text · Article · Oct 2014 · Journal of Molecular Biology
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    • "As the major DNA-binding proteins in eukaryotic cells are histones, accessibility to the DSB would be nearly guaranteed if the region around the lesion was largely histone free during DNA repair (79). This is indeed the case during repair of the HO-mediated DSB at the MAT locus from which histones are displaced presumably due to the actions of chromatin remodeling factors (80,81). However, if excess free histones are present in the vicinity of the DNA lesion, they can potentially compete with repair proteins for binding to DNA repair sites, thereby inhibiting or slowing down the repair process. "
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    ABSTRACT: In eukaryotes, multiple genes encode histone proteins that package genomic deoxyribonucleic acid (DNA) and regulate its accessibility. Because of their positive charge, ‘free’ (non-chromatin associated) histones can bind non-specifically to the negatively charged DNA and affect its metabolism, including DNA repair. We have investigated the effect of altering histone dosage on DNA repair in budding yeast. An increase in histone gene dosage resulted in enhanced DNA damage sensitivity, whereas deletion of a H3–H4 gene pair resulted in reduced levels of free H3 and H4 concomitant with resistance to DNA damaging agents, even in mutants defective in the DNA damage checkpoint. Studies involving the repair of a HO endonuclease-mediated DNA double-strand break (DSB) at the MAT locus show enhanced repair efficiency by the homologous recombination (HR) pathway on a reduction in histone dosage. Cells with reduced histone dosage experience greater histone loss around a DSB, whereas the recruitment of HR factors is concomitantly enhanced. Further, free histones compete with the HR machinery for binding to DNA and associate with certain HR factors, potentially interfering with HR-mediated repair. Our findings may have important implications for DNA repair, genomic stability, carcinogenesis and aging in human cells that have dozens of histone genes.
    Full-text · Article · Jul 2012 · Nucleic Acids Research
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