Chromatin remodeling and repair of DNA double-strand breaks

Eukaryotic cells have developed conserved mechanisms to efficiently sense and repair DNA damage that results from constant chromosomal lesions. DNA repair has to proceed in the context of chromatin, and both histone-modifiers and ATP-dependent chromatin remodelers have been implicated in this process. Here, we review the current understanding and new hypotheses on how different chromatin-modifying activities function in DNA repair in yeast and metazoan cells.     

Zinc finger protein 668 interacts with Tip60 to promote H2AX acetylation after DNA damage

Many tumor suppressors play an important role in the DNA damage pathway. Zinc finger protein 668 (ZNF668) has recently been identified as one of the potential tumor suppressors in breast cancer, but its function in DNA damage response is unknown. Herein, we report that ZNF668 is a regulator of DNA repair. ZNF668 knockdown impairs cell survival after DNA damage without affecting the ATM/ATR DNA-damage signaling cascade. However, recruitment of repair proteins to DNA lesions is decreased. In response to IR, ZNF668 knockdown reduces Tip60-H2AX interaction and impairs IR-induced histone H2AX hyperacetylation, thus impairing chromatin relaxation. Impaired chromatin relaxation causes decreased recruitment of repair proteins to DNA lesions, defective homologous recombination (HR) repair and impaired cell survival after IR. In addition, ZNF668 knockdown decreased RPA phosphorylation and its recruitment to DNA damage foci in response to UV. In both IR and UV damage responses, chromatin relaxation counteracted the impaired loading of repair proteins and DNA repair defects in ZNF668-deficient U2OS cells, indicating that impeded chromatin accessibility at sites of DNA breaks caused the DNA repair defects observed in the absence of ZNF668. Our findings suggest that ZNF668 is a key molecule that links chromatin relaxation with DNA damage response in DNA repair control.     

RecQ and RecJ process blocked replication forks prior to the resumption of replication in UV-irradiated Escherichia coli

The accurate recovery of replication following DNA damage and repair is critical for the maintenance of genomic integrity. In Escherichia coli, the recovery of replication following UV-induced DNA damage is dependent upon several proteins in the recF pathway, including RecF, RecO, and RecR. Two other recF pathway proteins, the RecQ helicase and the RecJ exonuclease, have been shown to affect the sites and frequencies at which illegitimate rearrangements occur following UV-induced DNA damage, suggesting that they also may function during the recovery of replication. We show here that RecQ and RecJ process the nascent DNA at blocked replication forks prior to the resumption of DNA synthesis. The processing involves selective degradation of the nascent lagging DNA strand and it requires both RecQ and RecJ. We suggest that this processing may serve to lengthen the substrate that can be recognized and stabilized by the RecA protein at the replication fork, thereby helping to ensure the accurate recovery of replication after the obstructing lesion has been repaired. Received: 1 June 1999 / Accepted: 28 July 1999     

Roles of chromatin remodellers in DNA double strand break repair

Now that we have a good understanding of the DNA double strand break (DSB) repair mechanisms and DSB-induced damage signalling, attention is focusing on the changes to the chromatin environment needed for efficient DSB repair. Mutations in chromatin remodelling complexes have been identified in cancers, making it important to evaluate how they impact upon genomic stability. Our current understanding of the DSB repair pathways suggests that each one has distinct requirements for chromatin remodelling. Moreover, restricting the extent of chromatin modifications could be a significant factor regulating the decision of pathway usage. In this review, we evaluate the distinct DSB repair pathways for their potential need for chromatin remodelling and review the roles of ATP-driven chromatin remodellers in the pathways.     

The histone acetyltransferase Hat1 facilitates DNA damage repair and morphogenesis in Candida albicans

Chromatin assembly and remodelling is an important process during the repair of DNA damage in eukaryotic cells. Although newly synthesized histone H4 is acetylated prior to nuclear import and incorporation into chromatin during DNA damage repair, the precise role of acetylation in this process is poorly understood. Here, we identify the histone acetyltransferase 1 (Hat1) catalysing the conserved acetylation pattern of histone H4 preceding its chromatin deposition in the fungal pathogen Candida albicans. Surprisingly, Hat1 is required for efficient repair of not just exogenous but also endogenous DNA damage. Cells lacking Hat1 rapidly accumulate DNA damages and switch from yeast‐like to pseudohyphal growth. In addition, reduction of histone H4 mimics lack of Hat1, suggesting that inefficient H4 supply for deposition into chromatin is the key functional consequence of Hat1 deficiency. Thus, remarkably, we demonstrate that C. albicans is the first organism known to require histone H4 processing for endogenous DNA damage repair and morphogenesis. Strikingly, we also discover that hat1Δ/Δ cells are hypersusceptible to caspofungin due to intracellular reactive oxygen species induced by this drug. Hence, we propose that targeting this class of histone acetyltransferases in fungal pathogens may have potential in antifungal therapy.     

Nucleosome Dynamics as Modular Systems that Integrate DNA Damage and Repair

By some estimates, a eukaryotic cell must repair up to 10,000 DNA lesions per cell cycle to counteract endogenous sources of DNA damage. Exposure to environmental toxins, UV sources, or other radiations only increases this enormous number. Failure to repair such lesions can lead to a deleterious mutation rate, genomic instability, or cell death. The timely and efficient repair of eukaryotic DNA damage is further complicated by the realization that DNA lesions must be detected and repaired in the context of chromatin with its complex organization within the nucleus. Numerous studies have shown that chromatin packaging can inhibit nearly all repair pathways, and recent work has defined specific mechanisms that facilitate DNA repair within the chromatin context. In this review, we provide a broad overview of chromatin regulatory mechanisms, mainly at the nucleosomal level, and then focus on recent work that elucidates the role of chromatin structure in regulating the timely and efficient repair of DNA double-strand breaks (DSBs).Although we tend to worry the most about environmental sources of DNA damage (e.g., chemical agents, UV radiation, ionizing radiation), it seems likely that much of the DNA repair machinery has evolved to contend with DNA lesions generated by the by-products of cellular metabolism—reactive oxygen species, endogenous alkylating agents, and DNA single- and double-strand breaks resulting from collapsed DNA replication forks or from oxidative destruction of deoxyribose residues (Lindahl and Wood 1999; Lindahl 2000). To combat the diversity of DNA lesions, cells have evolved a complex DNA damage response (DDR) that can engage many different DNA repair pathways, including nucleotide excision repair (NER), base excision repair (BER), DNA mismatch repair (MMR), single-strand annealing (SSA), nonhomologous end joining (NHEJ), and homologous recombination (HR). In eukaryotic cells, each of these repair pathways function in the context of a nucleoprotein structure, chromatin, which can potentially occlude DNA lesions from the repair machinery, and thus can influence the efficiency of repair. Early studies that focused on the response to UV damage, led to the access/repair/restore (ARR) model for repair of DNA lesions in chromatin (Green and Almouzni 2002). A central theme of this model is that chromatin inhibits the repair process, and thus it must be disrupted before or during the repair process, and then chromatin structures must be faithfully restored at the conclusion. What has become clear in the past few years, however, is that chromatin organization also serves a positive role in the DDR, to “prime” DNA repair events, functioning as a regulatory/integration platform that ensures that DNA repair is coordinated with other cellular events (Fig. 1). Here we focus on the repair of DNA double-strand breaks (DSBs), centering on the various mechanisms that facilitate this essential repair event within a chromatin context with a particular emphasis on the nucleosomal level. We envision that the concepts and themes discussed here will also be pertinent to other repair pathways, as discussed in several recent reviews (Adam and Polo 2012; Czaja et al. 2012; Lans et al. 2012; Odell et al. 2013).Open in a separate windowFigure 1.Access/prime/repair/restore model for the role of chromatin in the DDR. Chromatin remodeling and histone modification enzymes regulate both the accessibility of the lesion to repair factors as well as providing a platform for signaling repair events to other cellular processes. See text for details.     

Beyond Repair Foci: Subnuclear Domains and the Cellular Response to DNA Damage

In the mid to late 1990's several groups identified DNA damage-dependent focal accumulations in nuclei of both DNA repair factors and the phosphorylated form of the histone variant H2A.X. The term "repair foci" has since been used to describe these protein accumulations. As a molecular marker for DNA damage, they have been immensely useful in the study of signal transduction pathways triggered by DNA damage while aiding in the identification of new factors involved in DNA repair. In spite of their importance, many other changes in the nuclear landscape correlate with DNA damage and repair processes. These include dramatic changes in chromatin ultrastructure and epigenetic modifications, which occur at the site of DNA breaks as well as globally throughout the nucleus. Besides chromatin, DNA damage also affects the dynamic behaviour, morphology and biochemical composition of various subnuclear domains, including the nucleolus, promyelocytic leukemia (PML) nuclear bodies and Cajal bodies. These changes in the nuclear landscape, the topic of this review, appear to be intimately linked to the cellular response to DNA damage and may prove as useful as repair foci in elucidating mechanisms of DNA repair.     

Histone marks: repairing DNA breaks within the context of chromatin

Inherited or acquired defects in detecting, signalling or repairing DNA damage are associated with various human pathologies, including immunodeficiencies, neurodegenerative diseases and various forms of cancer. Nuclear DNA is packaged into chromatin and therefore the true in vivo substrate of damaged DNA occurs within the context of chromatin. Our work aims to decipher the mechanisms by which cells detect DNA damage and signal its presence to the DNA-repair and cell-cycle machineries. In particular, much of our work has focused on DNA DSBs (double-strand breaks) that are generated by ionizing radiation and radiomimetic chemicals, and which can also arise when the DNA replication apparatus encounters other DNA lesions. In the present review, we describe some of our recent work, as well as the work of other laboratories, that has identified new chromatin proteins that mediate DSB responses, control SDB processing or modulate chromatin structure at DNA-damage sites. We also aim to survey several recent advances in the field that have contributed to our understanding of how particular histone modifications and involved in DNA repair. It is our hope that by understanding the role of chromatin and its modifications in promoting DNA repair and genome stability, this knowledge will provide opportunities for developing novel classes of drugs to treat human diseases, including cancer.     

Double strand break (DSB) repair in heterochromatin and heterochromatin proteins in DSB repair

Chromosomal translocations are a hallmark of cancer cells and they represent a major cause of tumorigenesis. To avoid chromosomal translocations, faithful repair of DNA double strand breaks (DSBs) has to be ensured in the context of high ordered chromatin structure. However, chromatin compaction is proposed to represent a barrier for DSB repair. Here we review the different mechanisms cells use to alleviate the heterochromatic barrier for DNA repair. At the same time, we discuss the activating role of heterochromatin-associated proteins in this process, therefore proposing that chromatin structure, more than being a simple barrier, is a key modulator of DNA repair.     

Tilting at windmills? The nucleotide excision repair of chromosomal DNA

A typical view of how DNA repair functions in chromatin usually depicts a struggle in which the DNA repair machinery battles to overcome the inhibitory effect of chromatin on the repair process. It may be that in this current interpretation the repair mechanisms are ‘tilting at windmills’, fighting an imaginary foe. An emerging picture suggests that we should not consider chromatin as an inhibitory force to be overcome like some quixotic giant by the DNA repair processes. Instead we should now recognize that DNA repair and chromatin metabolism are inextricably and mechanistically linked. Here we discuss the latest findings which are beginning to reveal how changes in chromatin dynamics integrate with the DNA repair process in response to UV induced DNA damage, with an emphasis on events in the yeast Saccharomyces cerevisiae.     

Defective ATM‐Kap‐1‐mediated chromatin remodeling impairs DNA repair and accelerates senescence in progeria mouse model

ATM‐mediated phosphorylation of KAP‐1 triggers chromatin remodeling and facilitates the loading and retention of repair proteins at DNA lesions. Mouse embryonic fibroblasts (MEFs) derived from Zmpste24^?/? mice undergo early senescence, attributable to delayed recruitment of DNA repair proteins. Here, we show that ATM‐Kap‐1 signaling is compromised in Zmpste24^?/? MEFs, leading to defective DNA damage‐induced chromatin remodeling. Knocking down Kap‐1 rescues impaired chromatin remodeling, defective DNA repair and early senescence in Zmpste24^?/? MEFs. Thus, ATM‐Kap‐1‐mediated chromatin remodeling plays a critical role in premature aging, carrying significant implications for progeria therapy.