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.
"Studies in Arabidopsis thaliana implicate INO80-C in ectopic (transposon) recombination events , and arp8-depleted diploid yeast cells were modestly defective in interhomolog DSB repair by HR . 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 . Thus, it was argued that INO80-C contributes both to very early and very late stages of HR. "
[Show abstract][Hide abstract] 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.
"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. "
[Show abstract][Hide abstract] 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.
Nucleic Acids Research 07/2012; 40(19):9604-20. DOI:10.1093/nar/gks722 · 9.11 Impact Factor
"Multiple remodeling activities of INO80 family complexes have been reported to regulate different steps of the DSB-induced DDR. In yeast, the INO80 complex is recruited to DSBs in a H2A-phosphorylation-dependent manner and is required for removal of nucleosomes at DSB sites, affecting both HR and NHEJ [65,105-111]. Likewise, the INO80-related SWR1 complex is also recruited to DSBs in a H2A-phosphorylation-dependent manner, where it may function in nucleosome remodeling to regulate DSB repair [109,112,113]. "
[Show abstract][Hide abstract] ABSTRACT: The integrity of DNA is continuously challenged by metabolism-derived and environmental genotoxic agents that cause a variety of DNA lesions, including base alterations and breaks. DNA damage interferes with vital processes such as transcription and replication, and if not repaired properly, can ultimately lead to premature aging and cancer. Multiple DNA pathways signaling for DNA repair and DNA damage collectively safeguard the integrity of DNA. Chromatin plays a pivotal role in regulating DNA-associated processes, and is itself subject to regulation by the DNA-damage response. Chromatin influences access to DNA, and often serves as a docking or signaling site for repair and signaling proteins. Its structure can be adapted by post-translational histone modifications and nucleosome remodeling, catalyzed by the activity of ATP-dependent chromatin-remodeling complexes. In recent years, accumulating evidence has suggested that ATP-dependent chromatin-remodeling complexes play important, although poorly characterized, roles in facilitating the effectiveness of the DNA-damage response. In this review, we summarize the current knowledge on the involvement of ATP-dependent chromatin remodeling in three major DNA repair pathways: nucleotide excision repair, homologous recombination, and non-homologous end-joining. This shows that a surprisingly large number of different remodeling complexes display pleiotropic functions during different stages of the DNA-damage response. Moreover, several complexes seem to have multiple functions, and are implicated in various mechanistically distinct repair pathways.
E Hedström, C Pederiva, J Farnebo, B Nodin, K Jirström, D J Brennan, M Farnebo
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