The Chromatin Landscape around DNA Double-Strand Breaks in Yeast and Its Influence on DNA Repair Pathway Choice (original) (raw)
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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.
Mutations in two Ku homologs define a DNA end-joining repair pathway in Saccharomyces cerevisiae
Molecular and cellular biology, 1996
DNA double-strand break (DSB) repair in mammalian cells is dependent on the Ku DNA binding protein complex. However, the mechanism of Ku-mediated repair is not understood. We discovered a Saccharomyces cerevisiae gene (KU80) that is structurally similar to the 80-kDa mammalian Ku subunit. Ku8O associates with the product of the HDF1 gene, forming the major DNA end-binding complex of yeast cells. DNA end binding was absent in ku80delta, hdf1delta, or ku80delta hdf1delta strains. Antisera specific for epitope tags on Ku80 and Hdf1 were used in supershift and immunodepletion experiments to show that both proteins are directly involved in DNA end binding. In vivo, the efficiency of two DNA end-joining processes were reduced >10-fold in ku8Odelta, hdfldelta, or ku80delta hdf1delta strains: repair of linear plasmid DNA and repair of an HO endonuclease-induced chromosomal DSB. These DNA-joining defects correlated with DNA damage sensitivity, because ku80delta and hdf1delta strains were ...
Recruitment of the Recombinational Repair Machinery to a DNA Double-Strand Break in Yeast
Molecular Cell, 2003
viewed in Pâ ques and Haber, 1999; Sung et al., 2000). In the mouse, a homozygous null allele of RAD51 leads to embryonic lethality (Tsuzuki et al., 1996), and muta-Program in Molecular Medicine tions in RAD genes are associated with a spectrum of University of Massachusetts Medical School diseases, including cancer (reviewed in Ivanov and Ha-Worcester, Massachusetts 01605 ber, 1997; Jasin, 2000; Michelson and Weinert, 2000). 2 Institute of Biotechnology and Studies in yeast have suggested a sequence of molec-Department of Molecular Medicine ular events that occur following formation of a DSB (re-. First, the 5Ј ends of DNA that flank San Antonio, Texas 78245 the break are resected by an exonuclease. Rad51p, a functional homolog of the E. coli RecA recombinase, then binds the exposed single-stranded tails forming a right-Summary handed helical nucleoprotein filament. In vitro, Rad52p (Sung, 1997a) and a Rad55p/Rad57p heterodimer (Sung, Repair of DNA double-strand breaks (DSBs) by homol-1997b) can promote this early step by overcoming the ogous recombination requires members of the RAD52 inhibitory effects of the heterotrimeric single-stranded epistasis group. Here we use chromatin immunopre-DNA binding protein, RPA. The Rad51p nucleoprotein cipitation (ChIP) to examine the temporal order of filament is then believed to function in cooperation with recruitment of Rad51p, Rad52p, Rad54p, Rad55p, Rad54p to search the genome for a homologous pairing and RPA to a single, induced DSB in yeast. Our results partner and to form a heteroduplex "joint molecule" (Petsuggest a sequential, interdependent assembly of ukhova et al., 1998, 2000). Joint molecule formation is Rad proteins adjacent to the DSB initiated by binding followed by extension of the incoming strand by DNA of Rad51p. ChIP time courses from various mutant polymerases and branch migration, ultimately leading strains and additional biochemical studies suggest to restoration of the genetic information spanning the that Rad52p, Rad55p, and Rad54p each help promote break (reviewed in Pâ ques and Haber, 1999). the formation and/or stabilization of the Rad51p nu-Much less is known about how Rad proteins functioncleoprotein filament. We also find that all four Rad ally cooperate during DSB repair in vivo. Immunofluoresproteins associate with homologous donor sequences cence studies have shown that Rad51p, Rad52p, and during strand invasion. These studies provide a near Rad54p colocalize to "foci" in response to DNA damage comprehensive view of the molecular events required in vivo (Haaf et al., 1995; Tan et al., 1999), suggesting for the in vivo assembly of a functional Rad51p presynthat Rad proteins might function together within a larger, aptic filament. multiprotein complex. Consistent with this view, coimmunoprecipitation and yeast two-hybrid assays have Introduction shown that many members of the RAD52 group can interact with each other (Golub et al., 1997; Hays et al., DNA double-strand breaks (DSBs) arise in DNA due to 1995; Johnson and Symington, 1995; Krejci et al., 2001). environmental insults such as ionizing radiation or In contrast, recent studies indicate that the composition chemical exposure. DSBs also play an important role as of the damage-induced foci are dynamic, and photointermediates in DNA replication, immunoglobulin V(D)J bleaching studies indicate that several Rad proteins recombination, meiotic and mitotic crossing-over, and have very different diffusion coefficients, suggesting that yeast mating-type switching. Failure to correctly prothey may not exist together in a preassembled protein cess these DSBs can result in deletion or insertion of complex (Essers et al., 2002). genetic information, chromosomal fragmentation, trans-We wished to dissect how Rad proteins are recruited location, and chromosome loss. and function at a DSB in vivo. Here we use chromatin Homologous recombination (HR) is a major pathway immunoprecipitation (ChIP) analyses to examine the of DSB repair in all eukaryotes and has a distinct advantemporal order of Rad protein recruitment to a single, tage over other mechanisms in that it is mostly error induced DSB in yeast. Our results suggest a sequential free. Repair of DSBs by HR requires the RAD52 epistasis pathway, where Rad51p binds first, followed by Rad52p, group, defined by the yeast RAD50, RAD51, RAD52, Rad55p, and finally Rad54p. Each of these Rad proteins RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2 genes. also associates with the homologous donor sequences These genes are highly conserved among all eukaryotes during strand invasion. We further examined the func-(Cromie et al., 2001; Pâ ques and Haber, 1999; Sung et tional interdependencies among these proteins by peral., 2000), highlighting the importance of these proteins
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 .
Methods, 2009
DNA repair occurs in a chromatin context, and nucleosome remodeling is now recognized as an important regulatory feature by allowing repair factors access to damaged sites. The yeast mating type locus (MAT) has emerged an excellent model to study the role of chromatin remodeling at a well-defined DNA double-strand break (DSB). We discuss methods to study nucleosome dynamics and DSB repair factor recruitment to the MAT locus after a DSB has been formed.
F1000 - Post-publication peer review of the biomedical literature
Nonhomologous end joining (NHEJ) is an important DNA double-strand-break (DSB) repair pathway that requires three protein complexes in Saccharomyces cerevisiae: the Ku heterodimer (Yku70-Yku80), MRX (Mre11-Rad50-Xrs2), and DNA ligase IV (Dnl4-Lif1), as well as the ligase-associated protein Nej1. Here we use chromatin immunoprecipitation from yeast to dissect the recruitment and release of these protein complexes at HO-endonuclease-induced DSBs undergoing productive NHEJ. Results revealed that Ku and MRX assembled at a DSB independently and rapidly after DSB formation. Ligase IV appeared at the DSB later than Ku and MRX and in a strongly Ku-dependent manner. Ligase binding was extensive but slightly delayed in rad50 yeast. Ligase IV binding occurred independently of Nej1, but instead promoted loading of Nej1. Interestingly, dissociation of Ku and ligase from unrepaired DSBs depended on the presence of an intact MRX complex and ATP binding by Rad50, suggesting a possible role of MRX in terminating a NHEJ repair phase. This activity correlated with extended DSB resection, but limited degradation of DSB ends occurred even in MRX mutants with persistently bound Ku. These findings reveal the in vivo assembly of the NHEJ repair complex and shed light on the mechanisms controlling DSB repair pathway utilization.
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.
DNA Repair, 2015
DNA double strand breaks (DSB)s often require end processing prior to joining during their repair by non-homologous end joining (NHEJ). Although the yeast proteins, Pol4, a Pol X family DNA polymerase, and Rad27, a nuclease, participate in the end processing reactions of NHEJ, the mechanisms underlying the recruitment of these factors to DSBs are not known. Here we demonstrate that Nej1, a NHEJ factor that interacts with and modulates the activity of the NHEJ DNA ligase complex (Dnl4/Lif1), physically and functionally interacts with both Pol4 and Rad27. Notably, Nej1 and Dnl4/Lif1, which also interacts with both Pol4 and Rad27, independently recruit the end processing factors to in vivo DSBs via mechanisms that are additive rather than redundant. As was observed with Dnl4/Lif1, the activities of both Pol4 and Rad27 were enhanced by the interaction with Nej1. Furthermore, Nej1 increased the joining of incompatible DNA ends in reconstituted reactions containing Pol4, Rad27 and Dnl4/Lif1, indicating that the stimulatory activities of Nej1 and Dnl4/Lif1 are also additive. Together our results reveal novel roles for Nej1 in the recruitment of Pol4 and Rad27 to in vivo DSBs and the coordination of the end processing and ligation reactions of NHEJ.
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.
Molecular and cellular biology, 1997
During repair of a double-strand break (DSB) by gene conversion, one or both 3' ends of the DSB invade a homologous donor sequence and initiate new DNA synthesis. The use of the invading DNA strand as a primer for new DNA synthesis requires that any nonhomologous bases at the 3' end be removed. We have previously shown that removal of a 3' nonhomologous tail in Saccharomyces cerevisiae depends on the nucleotide excision repair endonuclease Rad1/Rad10, and also on the mismatch repair proteins Msh2 and Msh3. We now report that these four proteins are needed only when the nonhomologous ends of recombining DNA are 30 nucleotides (nt) long or longer. An additional protein, the helicase Srs2, is required for the RAD1-dependent removal of long 3' tails. We suggest that Srs2 acts to extend and stabilize the initial nascent joint between the invading single strand and its homolog. 3' tails shorter than 30 nt are removed by another mechanism that depends at least in part o...