Chromatin dynamics after DNA damage: The legacy of the access–repair–restore model

Eukaryotic genomes are packaged into chromatin, which is the physiological substrate for all DNA transactions, including DNA damage and repair. Chromatin organization imposes major constraints on DNA damage repair and thus undergoes critical rearrangements during the repair process. These rearrangements have been integrated into the “access–repair–restore” (ARR) model, which provides a molecular framework for chromatin dynamics in response to DNA damage. Here, we take a historical perspective on the elaboration of this model and describe the molecular players involved in damaged chromatin reorganization in human cells. In particular, we present our current knowledge of chromatin assembly coupled to DNA damage repair, focusing on the role of histone variants and their dedicated chaperones. Finally, we discuss the impact of chromatin rearrangements after DNA damage on chromatin function and epigenome maintenance.     

Chromatin modulation and the DNA damage response

The ability to sense and respond appropriately to genetic lesions is vitally important to maintain the integrity of the genome. Emerging evidence indicates that various modulations to chromatin structure are centrally important to many aspects of the DNA damage response (DDR). Here, we discuss recently described roles for specific post-translational covalent modifications to histone proteins, as well as ATP-dependent chromatin remodelling, in DNA damage signalling and repair of DNA double strand breaks.     

RAD54 forms DNA repair foci in response to DNA damage in living plant cells

Plants have various defense mechanisms against environmental stresses that induce DNA damage. Genetic and biochemical analyses have revealed the sensing and signaling of DNA damage, but little is known about subnuclear dynamics in response to DNA damage in living plant cells. Here, we observed that the chromatin remodeling factor RAD54, which is involved in DNA repair via the homologous recombination pathway, formed subnuclear foci (termed RAD54 foci) in Arabidopsis thaliana after induction of DNA double‐strand breaks. The appearance of RAD54 foci was dependent on the ATAXIA‐TELANGIECTASIA MUTATED–SUPPRESSOR OF GAMMA RESPONSE 1 pathway, and RAD54 foci were co‐localized with γH2AX signals. Laser irradiation of a subnuclear area demonstrated that in living cells RAD54 was specifically accumulated at the damaged site. In addition, the formation of RAD54 foci showed specificity for cell type and region. We conclude that RAD54 foci correspond to DNA repair foci in A. thaliana.     

Chromatin structure in double strand break repair

Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA double strand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a result of chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizing radiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs are associated with oxidative metabolism, form during the normal S phase, when replication forks collapse and are generated during physiological processes such as V(D)J recombination, yeast mating type switching and meiosis. It is estimated that in mammalian cells ∼10 DSBs per cell are formed daily. If left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellular response to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore the structure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumental to the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repair and signaling molecules to the lesion. Here, we review the key findings related to each of these functions and examine their cross-talk.     

Preserving genome integrity and function: the DNA damage response and histone modifications

Modulation of chromatin templates in response to cellular cues, including DNA damage, relies heavily on the post-translation modification of histones. Numerous types of histone modifications including phosphorylation, methylation, acetylation, and ubiquitylation occur on specific histone residues in response to DNA damage. These histone marks regulate both the structure and function of chromatin, allowing for the transition between chromatin states that function in undamaged condition to those that occur in the presence of DNA damage. Histone modifications play well-recognized roles in sensing, processing, and repairing damaged DNA to ensure the integrity of genetic information and cellular homeostasis. This review highlights our current understanding of histone modifications as they relate to DNA damage responses (DDRs) and their involvement in genome maintenance, including the potential targeting of histone modification regulators in cancer, a disease that exhibits both epigenetic dysregulation and intrinsic DNA damage.     

Accessing DNA damage in chromatin: Preparing the chromatin landscape for base excision repair

DNA damage in chromatin comes in many forms, including single base lesions that induce base excision repair (BER). We and others have shown that the structural location of DNA lesions within nucleosomes greatly influences their accessibility to repair enzymes. Indeed, a difference in the location of uracil as small as one-half turn of the DNA backbone on the histone surface can result in a 10-fold difference in the time course of its removal in vitro. In addition, the cell has evolved several interdependent processes capable of enhancing the accessibility of excision repair enzymes to DNA lesions in nucleosomes, including post-translational modification of histones, ATP-dependent chromatin remodeling and interchange of histone variants in nucleosomes. In this review, we focus on different factors that affect accessibility of BER enzymes to nucleosomal DNA.     

Role of deubiquitinases in DNA damage response

DNA damage response (DDR) serves as an integrated cellular network to detect cellular stress and react by activating pathways responsible for halting cell cycle progression, stimulating DNA damage repair, and initiating apoptosis. Efficient DDR protects cells from genomic instability while defective DDR can allow DNA lesions to go unrepaired, causing permanent mutations that will affect future generations of cells and possibly cause disease conditions such as cancer. Therefore, DDR mechanisms must be tightly regulated in order to ensure organismal health and viability. One major way of DDR regulation is ubiquitination, which has been long known to control DDR protein localization, activity, and stability. The reversal of this process, deubiquitination, has more recently come to the forefront of DDR research as an important new angle in ubiquitin-mediated regulation of DDR. As such, deubiquitinases have emerged as key factors in DDR. Importantly, deubiquitinases are attractive small-molecule drug targets due to their well-defined catalytic residues that provide a promising avenue for developing new cancer therapeutics. This review focuses on the emerging roles of deubiquitinases in various DNA repair pathways.     

Monoubiquitination of H2AX protein regulates DNA damage response signaling

Double strand breaks (DSBs) are the most deleterious of the DNA lesions that initiate genomic instability and promote tumorigenesis. Cells have evolved a complex protein network to detect, signal, and repair DSBs. In mammalian cells, a key component in this network is H2AX, which becomes rapidly phosphorylated at Ser(139) (γ-H2AX) at DSBs. Here we show that monoubiquitination of H2AX mediated by the RNF2-BMI1 complex is critical for the efficient formation of γ-H2AX and functions as a proximal regulator in DDR (DNA damage response). RNF2-BMI1 interacts with H2AX in a DNA damage-dependent manner and is required for monoubiquitination of H2AX at Lys(119)/Lys(120). As a functional consequence, we show that the H2AX K120R mutant abolishes H2AX monoubiquitination, impairs the recruitment of p-ATM (Ser(1981)) to DSBs, and thereby reduces the formation of γ-H2AX and the recruitment of MDC1 to DNA damage sites. These data suggest that monoubiquitination of H2AX plays a critical role in initiating DNA damage signaling. Consistent with these observations, impairment of RNF2-BMI1 function by siRNA knockdown or overexpression of the ligase-dead RNF2 mutant all leads to significant defects both in accumulation of γ-H2AX, p-ATM, and MDC1 at DSBs and in activation of NBS1 and CHK2. Additionally, the regulatory effect of RNF2-BMI1 on γ-H2AX formation is dependent on ATM. Lacking their ability to properly activate the DNA damage signaling pathway, RNF2-BMI1 complex-depleted cells exhibit impaired DNA repair and increased sensitivity to ionizing radiation. Together, our findings demonstrate a distinct monoubiquitination-dependent mechanism that is required for H2AX phosphorylation and the initiation of DDR.     

DNA damage response and repair in ovarian cancer: Potential targets for therapeutic strategies

Ovarian cancer is among the most lethal gynecologic malignancies with a poor survival prognosis. The current therapeutic strategies involve surgery and chemotherapy. Research is now focused on novel agents especially those targeting DNA damage response (DDR) pathways. Understanding the DDR process in ovarian cancer necessitates having a detailed knowledge on a series of signaling mediators at the cellular and molecular levels. The complexity of the DDR process in ovarian cancer and how this process works in metastatic conditions is comprehensively reviewed. For evaluating the efficacy of therapeutic agents targeting DNA damage in ovarian cancer, we will discuss the components of this system including DDR sensors, DDR transducers, DDR mediators, and DDR effectors. The constituent pathways include DNA repair machinery, cell cycle checkpoints, and apoptotic pathways. We also will assess the potential of active mediators involved in the DDR process such as therapeutic and prognostic candidates that may facilitate future studies.     

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.     

Uhrf2 is important for DNA damage response in vascular smooth muscle cells

Emerging evidence shows that Uhrf1 plays an important role in DNA damage response for maintaining genomic stability. Interestingly, Uhrf1 has a paralog Uhrf2 in mammals. Uhrf1 and Uhrf2 share similar domain architectures. However, the role of Uhrf2 in DNA damage response has not been studied yet. During the analysis of the expression level of Uhrf2 in different tissues, we found that Uhrf2 is highly expressed in aorta and aortic vascular smooth muscle cells. Thus, we studied the role of Uhrf2 in DNA damage response in aortic vascular smooth muscle cells. Using laser microirradiation, we found that like Uhrf1, Uhrf2 was recruited to the sites of DNA damage. We dissected the functional domains of Uhrf2 and found that the TTD, PHD and SRA domains are important for the relocation of Uhrf2 to the sites of DNA damage. Moreover, depletion of Uhrf2 suppressed DNA damage-induced H2AX phosphorylation and DNA damage repair. Taken together, our results demonstrate the function of Uhrf2 in DNA damage response.     

Ubiquitin and the DNA damage response

Comment on: Gatti M, et al. Cell Cycle 2012; 11:2538-44.     

Chromatin stiffening underlies enhanced locus mobility after DNA damage in budding yeast

DNA double‐strand breaks (DSBs) induce a cellular response that involves histone modifications and chromatin remodeling at the damaged site and increases chromosome dynamics both locally at the damaged site and globally in the nucleus. In parallel, it has become clear that the spatial organization and dynamics of chromosomes can be largely explained by the statistical properties of tethered, but randomly moving, polymer chains, characterized mainly by their rigidity and compaction. How these properties of chromatin are affected during DNA damage remains, however, unclear. Here, we use live cell microscopy to track chromatin loci and measure distances between loci on yeast chromosome IV in thousands of cells, in the presence or absence of genotoxic stress. We confirm that DSBs result in enhanced chromatin subdiffusion and show that intrachromosomal distances increase with DNA damage all along the chromosome. Our data can be explained by an increase in chromatin rigidity, but not by chromatin decondensation or centromeric untethering only. We provide evidence that chromatin stiffening is mediated in part by histone H2A phosphorylation. Our results support a genome‐wide stiffening of the chromatin fiber as a consequence of DNA damage and as a novel mechanism underlying increased chromatin mobility.     

Crosstalk between P53 and DNA damage response in ageing

Ageing is a sophisticated process, accompanied by reduction in general physiological capacity and increase in mortality and death, stemming from damage accumulation over time. Various signaling pathways are known to be involved in the functional decrease in various organs in ageing humans. One of the most prominent pathways is DNA damage response (DDR), which is responsible for maintenance of the genomic integrity and stability. Insufficient or dysfunctional DDR signaling and the subsequent accumulation of potential DNA lesions are associated with the initiation/progression of various human pathologies including ageing. As a tumor suppressor gene, with critical functions in the ageing process, p53 is considered as a DDR centerpiece. In this review, we aim to discuss the interactions between p53 and DDR signaling and their contributions in ageing.