Haico van Attikum

Recruitment of the INO80 Complex by H2A Phosphorylation Links ATP-Dependent Chromatin Remodeling with DNA Double-Strand Break Repair

The budding yeast INO80 complex is a conserved ATP-dependent nucleosome remodeler containing actin-related proteins Arp5 and Arp8. Strains lacking INO80, ARP5, or ARP8 have defects in transcription. Here we show that these mutants are hypersensitive to DNA damaging agents and to double-strand breaks (DSBs) induced by the HO endonuclease. The checkpoint response and most transcriptional modulation associated with induction of DNA damage are unaffected by these mutations. Using chromatin immunoprecipitation we show that Ino80, Arp5, and Arp8 are recruited to an HO-induced DSB, where a phosphorylated form of H2A accumulates. Recruitment of Ino80 is compromised in cells lacking the H2A phosphoacceptor S129. Finally, we demonstrate that conversion of the DSB into ssDNA is compromised in arp8 and H2A mutants, which are both deficient for INO80 activity at the site of damage. These results implicate INO80-mediated chromatin remodeling directly at DSBs, where it appears to facilitate processing of the lesion.

Crosstalk between histone modifications during the DNA damage response

Chromatin structure has a crucial role in processes of metabolism, including transcription, DNA replication and DNA damage repair. An evolutionarily conserved variant of histone H2A, called H2AX, is one of the key components of chromatin. H2AX becomes rapidly phosphorylated on chromatin surrounding DNA double-strand breaks (DSBs). Recent studies have shown that H2AX and other components of damaged chromatin also become modified by acetylation and ubiquitylation. This review discusses how specific combinations of histone modifications affect the accumulation and function of DNA repair factors (MDC1, RNF8, RNF168, 53BP1, BRCA1) and chromatin remodeling complexes (INO80, SWR1, TIP60-p400) at DSBs. These collectively regulate DSB repair and checkpoint arrest, avoiding genomic instability and oncogenic transformation in higher eukaryotes.

Rewiring of Genetic Networks in Response to DNA Damage

DNA Damage Pathways Revealed Despite the dynamic nature of cellular responses, the genetic networks that govern these responses have been mapped primarily as static snapshots. Bandyopadhyay et al. (p. 1385; see the Perspective by Friedman and Schuldiner) report a comparison of large genetic interactomes measured among all yeast kinases, phosphatases, and transcription factors, as the cell responded to DNA damage. The interactomes revealed were highly dynamic structures that changed dramatically with changing conditions. These dynamic interactions reveal genetic relationships that can be more effective than classical “static” interactions (for example, synthetic lethals and epistasis maps) in identifying pathways of interest. A network comparison of genetic interactions mapped at two conditions reveals genetic responses to DNA damage in yeast. Although cellular behaviors are dynamic, the networks that govern these behaviors have been mapped primarily as static snapshots. Using an approach called differential epistasis mapping, we have discovered widespread changes in genetic interaction among yeast kinases, phosphatases, and transcription factors as the cell responds to DNA damage. Differential interactions uncover many gene functions that go undetected in static conditions. They are very effective at identifying DNA repair pathways, highlighting new damage-dependent roles for the Slt2 kinase, Pph3 phosphatase, and histone variant Htz1. The data also reveal that protein complexes are generally stable in response to perturbation, but the functional relations between these complexes are substantially reorganized. Differential networks chart a new type of genetic landscape that is invaluable for mapping cellular responses to stimuli.

The histone code at DNA breaks: a guide to repair?

Chromatin modifications are important for all cellular processes that involve DNA, including transcription, replication and DNA repair. Chromatin can be modified by the addition of adducts to histone tail residues or by nucleosome remodelling, which requires ATP-dependent chromatin-remodelling complexes. Although the role of these mechanisms in transcription is well studied, their impact on DNA repair has only recently become evident. One crucial chromatin modification, the phosphorylation of histone H2A, links the recruitment of histone modifiers and ATP-dependent chromatin-remodelling complexes to sites of DNA damage.

Distinct roles for SWR1 and INO80 chromatin remodeling complexes at chromosomal double-strand breaks.

INO80 and SWR1 are two closely related ATP‐dependent chromatin remodeling complexes that share several subunits. Ino80 was reported to be recruited to the HO endonuclease‐induced double‐strand break (DSB) at the budding yeast mating‐type locus, MAT. We find Swr1 similarly recruited in a manner dependent on the phosphorylation of H2A (γH2AX). This is not unique to cleavage at MAT; both Swr1 and Ino80 bind near an induced DSB on chromosome XV. Whereas Swr1 incorporates the histone variant H2A.Z into chromatin at promoters, H2A.Z levels do not increase at DSBs. Instead, H2A.Z, γH2AX and core histones are coordinately removed near the break in an INO80‐dependent, but SWR1‐independent, manner. Mutations in INO80‐specific subunits Arp8 or Nhp10 impair the binding of Mre11 nuclease, yKu80 and ATR‐related Mec1 kinase at the DSB, resulting in defective end‐processing and checkpoint activation. In contrast, Mre11 binding, end‐resection and checkpoint activation were normal in the swr1 strain, but yKu80 loading and error‐free end‐joining were impaired. Thus, these two related chromatin remodelers have distinct roles in DSB repair and checkpoint activation.

The NuRD chromatin–remodeling complex regulates signaling and repair of DNA damage

NuRD is recruited to DNA double-strand breaks, where it promotes RNF8/RNF168 histone ubiquitylation and accumulation of DNA repair factors (see also related paper by Larsen et al. in this issue).

The yeast Fun30 and human SMARCAD1 chromatin remodellers promote DNA end resection

Several homology-dependent pathways can repair potentially lethal DNA double-strand breaks (DSBs). The first step common to all homologous recombination reactions is the 5′–3′ degradation of DSB ends that yields the 3′ single-stranded DNA required for the loading of checkpoint and recombination proteins. In yeast, the Mre11–Rad50–Xrs2 complex (Xrs2 is known as NBN or NBS1 in humans) and Sae2 (known as RBBP8 or CTIP in humans) initiate end resection, whereas long-range resection depends on the exonuclease Exo1, or the helicase–topoisomerase complex Sgs1–Top3–Rmi1 together with the endonuclease Dna2 (refs 1–6). DSBs occur in the context of chromatin, but how the resection machinery navigates through nucleosomal DNA is a process that is not well understood. Here we show that the yeast Saccharomyces cerevisiae Fun30 protein and its human counterpart SMARCAD1 (ref. 8), two poorly characterized ATP-dependent chromatin remodellers of the Snf2 ATPase family, are directly involved in the DSB response. Fun30 physically associates with DSB ends and directly promotes both Exo1- and Sgs1-dependent end resection through a mechanism involving its ATPase activity. The function of Fun30 in resection facilitates the repair of camptothecin-induced DNA lesions, although it becomes dispensable when Exo1 is ectopically overexpressed. Interestingly, SMARCAD1 is also recruited to DSBs, and the kinetics of recruitment is similar to that of EXO1. The loss of SMARCAD1 impairs end resection and recombinational DNA repair, and renders cells hypersensitive to DNA damage resulting from camptothecin or poly(ADP-ribose) polymerase inhibitor treatments. These findings unveil an evolutionarily conserved role for the Fun30 and SMARCAD1 chromatin remodellers in controlling end resection, homologous recombination and genome stability in the context of chromatin.

Non-homologous end-joining proteins are required for Agrobacterium T-DNA integration

Agrobacterium tumefaciens causes crown gall disease in dicotyledonous plants by introducing a segment of DNA (T‐DNA), derived from its tumour‐inducing (Ti) plasmid, into plant cells at infection sites. Besides these natural hosts, Agrobacterium can deliver the T‐DNA also to monocotyledonous plants, yeasts and fungi. The T‐DNA integrates randomly into one of the chromosomes of the eukaryotic host by an unknown process. Here, we have used the yeast Saccharomyces cerevisiae as a T‐DNA recipient to demonstrate that the non‐homologous end‐joining (NHEJ) proteins Yku70, Rad50, Mre11, Xrs2, Lig4 and Sir4 are required for the integration of T‐DNA into the host genome. We discovered a minor pathway for T‐DNA integration at the telomeric regions, which is still operational in the absence of Rad50, Mre11 or Xrs2, but not in the absence of Yku70. T‐DNA integration at the telomeric regions in the rad50, mre11 and xrs2 mutants was accompanied by gross chromosomal rearrangements.

Chromatin and the DNA damage response: The cancer connection

The integrity of the human genome is constantly threatened by genotoxic agents that cause DNA damage. Inefficient or inaccurate repair of DNA lesions triggers genome instability and can lead to cancer development or even cell death. Cells counteract the adverse effects of DNA lesions by activating the DNA damage response (DDR), which entails a coordinated series of events that regulates cell cycle progression and repair of DNA lesions. Efficient DNA repair in living cells is complicated by the packaging of genomic DNA into a condensed, often inaccessible structure called chromatin. Cells utilize post‐translational histone modifications and ATP‐dependent chromatin remodeling to modulate chromatin structure and increase the accessibility of the repair machinery to lesions embedded in chromatin. Here we review and discuss our current knowledge and recent advances on DNA damage‐induced chromatin changes and their implications for the mammalian DNA damage response, genome stability and carcinogenesis. Exploiting our improving understanding of how modulators of chromatin structure orchestrate the DDR may provide new avenues to improve cancer management.

DDB2 promotes chromatin decondensation at UV-induced DNA damage

In addition to its role in DNA lesion recognition, the damaged DNA-binding protein DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex.