Analysis of chromatin remodeling during formation of a DNA double-strand break at the yeast mating type locus (original) (raw)
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Chromatin remodelling at a DNA double-strand break site in Saccharomyces cerevisiae
Nature, 2005
The repair of DNA double-strand breaks (DSBs) is critical for maintaining genome stability. Eukaryotic cells repair DSBs using both non-homologous end joining (NHEJ) and homologous recombination (HR). How chromatin structure is altered in response to DSBs and how such alterations influence DSB repair processes are important questions. In vertebrates, phosphorylation of the histone variant H2A.X (γ-H2A) occurs rapidly after formation of DSBs 1 , spreads over megabase chromatin domains, and is required for stable accumulation of DNA repair proteins at DNA damage foci 2 . In Saccharomyces cerevisiae, phosphorylation of the two major H2A species is also signaled by DSB formation, spreading ∼40 Kb in either direction from a DSB 3 . Here we show that near a DSB γ-H2A is followed by loss of histones H2B and H3 and increased sensitivity of chromatin to digestion by micrococcal nuclease. However, γ-H2A and nucleosome loss occur independently of one another. The DNA damage sensor MRX (Mre11-Rad50-Xrs2) 4 is required for histone eviction, which additionally depends on the ATP-dependent nucleosome-remodeling complex, INO80 5 . The repair protein Rad51 6 shows delayed recruitment to a DSB in the absence of histone loss, suggesting that MRX-dependent nucleosome remodeling regulates the accessibility of factors with direct roles in DNA damage repair by HR. We propose that MRX regulates two pathways of chromatin changes, including nucleosome displacement, required for efficient recruitment of HR proteins, and γ-H2A, which modulates checkpoint responses to DNA damage 2 .
International Journal of Molecular Sciences
DNA double-strand breaks (DSBs) are harmful DNA lesions, which elicit catastrophic consequences for genome stability if not properly repaired. DSBs can be repaired by either non-homologous end joining (NHEJ) or homologous recombination (HR). The choice between these two pathways depends on which proteins bind to the DSB ends and how their action is regulated. NHEJ initiates with the binding of the Ku complex to the DNA ends, while HR is initiated by the nucleolytic degradation of the 5′-ended DNA strands, which requires several DNA nucleases/helicases and generates single-stranded DNA overhangs. DSB repair occurs within a precisely organized chromatin environment, where the DNA is wrapped around histone octamers to form the nucleosomes. Nucleosomes impose a barrier to the DNA end processing and repair machinery. Chromatin organization around a DSB is modified to allow proper DSB repair either by the removal of entire nucleosomes, thanks to the action of chromatin remodeling factors,...
DNA Repair, 2010
a b s t r a c t DNA non-homologous end-joining (NHEJ) and homologous recombination (HR) represent the major DNA double strand break (DSB) pathways in mammalian cells, whilst ataxia telangiectasia mutated (ATM) lies at the core of the DSB signalling response. ATM signalling plays a major role in modifying chromatin structure in the vicinity of the DSB and increasing evidence suggests that this function influences the DSB rejoining process. DSBs have long been known to be repaired with two (or more) component kinetics. The majority (∼85%) of DSBs are repaired with fast kinetics in a predominantly ATM-independent manner. In contrast, ∼15% of radiation-induced DSBs are repaired with markedly slower kinetics via a process that requires ATM and those mediator proteins, such as MDC1 or 53BP1, that accumulate at ionising radiation induced foci (IRIF). DSBs repaired with slow kinetics predominantly localise to the periphery of genomic heterochromatin (HC). Indeed, there is mounting evidence that chromatin complexity and not damage complexity confers slow DSB repair kinetics. ATM's role in HC-DSB repair involves the direct phosphorylation of KAP-1, a key HC formation factor. KAP-1 phosphorylation (pKAP-1) arises in both a pan-nuclear and a focal manner after radiation and ATM-dependent pKAP-1 is essential for DSB repair within HC regions. Mediator proteins such as 53BP1, which are also essential for HC-DSB repair, are expendable for pan-nuclear pKAP-1 whilst being essential for pKAP-1 formation at IRIF. Data suggests that the essential function of the mediator proteins is to promote the retention of activated ATM at DSBs, concentrating the phosphorylation of KAP-1 at HC DSBs. DSBs arising in G2 phase are also repaired with fast and slow kinetics but, in contrast to G0/G1 where they all DSBs are repaired by NHEJ, the slow component of DSB repair in G2 phase represents an HR process involving the Artemis endonuclease. Results suggest that whilst NHEJ repairs the majority of DSBs in G2 phase, Artemis-dependent HR uniquely repairs HC DSBs. Collectively, these recent studies highlight not only how chromatin complexity influences the factors required for DSB repair but also the pathway choice.
Chromatin dynamics during repair of chromosomal DNA double-strand breaks
Epigenomics, 2009
The integrity of a eukaryotic genome is often challenged by DNA double-strand breaks (DSBs). Even a single, unrepaired DSB can be a lethal event, or such unrepaired damage can result in chromosomal instability and loss of genetic information. Furthermore, defects in the pathways that respond to and repair DSBs can lead to the onset of several human pathologic disorders with pleiotropic clinical features, including age-related diseases and cancer. For decades, studies have focused on elucidating the enzymatic mechanisms involved in recognizing, signaling and repairing DSBs within eukaryotic cells. The majority of biochemical and genetic studies have used simple, DNA substrates, whereas only recently efforts have been geared towards understanding how the repair machinery deals with DSBs within chromatin fibers, the nucleoprotein complex that packages DNA within the eukaryotic nucleus. The aim of this review is to discuss our recent understanding of the relationship between chromatin s...
When repair meets chromatin: First in series on chromatin dynamics
EMBO Reports, 2002
In eukaryotic cells, the inheritance of both the DNA sequence and its organization into chromatin is critical to maintain genome stability. This maintenance is challenged by DNA damage. To fully understand how the cell can tolerate genotoxic stress, it is necessary to integrate knowledge of the nature of DNA damage, its detection and its repair within the chromatin environment of a eukaryotic nucleus. The multiplicity of the DNA damage and repair processes, as well as the complex nature of chromatin, have made this issue difficult to tackle. Recent progress in each of these areas enables us to address, both at a molecular and a cellular level, the importance of interrelationships between them. In this review we revisit the 'access, repair, restore' model, which was proposed to explain how the conserved process of nucleotide excision repair operates within chromatin. Recent studies have identified factors potentially involved in this process and permit refinement of the basic model. Drawing on this model, the chromatin alterations likely to be required during other processes of DNA damage repair, particularly double-strand break repair, are discussed and recently identified candidates that might perform such alterations are highlighted.
The Yeast Chromatin Remodeler RSC Complex Facilitates End Joining Repair of DNA Double-Strand Breaks
Molecular and Cellular Biology, 2005
Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30⌬ maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.
DNA repair choice defines a common pathway for recruitment of chromatin regulators
Nature Communications, 2013
DNA double-strand break (DSB) repair is essential for maintenance of genome stability. Recent work has implicated a host of chromatin regulators in the DNA damage response, and although several functional roles have been defined, the mechanisms that control their recruitment to DNA lesions remain unclear. Here, we find that efficient DSB recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of γH2AX. Our work indicates that cell cycle position plays a key role in DNA repair pathway choice and that recruitment of chromatin regulators is tightly coupled to homologous recombination.
Chromatin remodeling in DNA double-strand break repair
Current Opinion in Genetics & Development, 2007
ATP-dependent chromatin remodeling complexes use ATP hydrolysis to remodel nucleosomes and have well-established functions in transcription. However, emerging lines of evidence suggest that chromatin remodeling complexes are important players in DNA double-strand break (DSB) repair as well. The INO80 and SWI2 subfamilies of chromatin remodeling complexes have been found to be recruited to the doublestrand lesions and to function directly in both homologous recombination and non-homologous end-joining, the two major conserved DSB repair pathways. Improperly repaired DSBs are implicated in cancer development in higher organisms. Understanding how chromatin remodeling complexes contribute to DSB repair should provide new insights into the mechanisms of carcinogenesis and might suggest new targets for cancer treatment.
Recombinational Repair within Heterochromatin Requires ATP-Dependent Chromatin Remodeling
Cell, 2009
Heterochromatin plays a key role in protection of chromosome integrity by suppressing homologous recombination. In Saccharomyces cerevisiae, Sir2p, Sir3p, and Sir4p are structural components of heterochromatin found at telomeres and the silent mating-type loci. Here we have investigated whether incorporation of Sir proteins into minichromosomes regulates early steps of recombinational repair in vitro. We find that addition of Sir3p to a nucleosomal substrate is sufficient to eliminate yRad51pcatalyzed formation of joints, and that this repression is enhanced by Sir2p/Sir4p. Importantly, Sir-mediated repression requires histone residues that are critical for silencing in vivo. Moreover, we demonstrate that the SWI/SNF chromatin-remodeling enzyme facilitates joint formation by evicting Sir3p, thereby promoting subsequent Rad54p-dependent formation of a strand invasion product. These results suggest that recombinational repair in the context of heterochromatin presents additional constraints that can be overcome by ATP-dependent chromatinremodeling enzymes.