AtMMS21 regulates DNA damage response and homologous recombination repair in Arabidopsis

DNA damage is a significant problem in living organisms and DNA repair pathways have been evolved in different species to maintain genomic stability. Here we demonstrated the molecular function of AtMMS21, a component of SMC5/6 complex, in plant DNA damage response. Compared with wild type, the AtMMS21 mutant plants show hypersensitivity in the DNA damaging treatments by MMS, cisplatin and gamma radiation. However, mms21-1 is not sensitive to replication blocking agents hydroxyurea and aphidicolin. The expression of a DNA damage response gene PARP2 is upregulated in mms21-1 under normal condition, suggesting that this signaling pathway is constitutively activated in the mutant. Depletion of ATAXIA-TELANGIECTASIA MUTATED (ATM) in mms21-1 enhances its root growth defect phenotype, indicating that ATM and AtMMS21 may play additive roles in DNA damage pathway. The analysis of homologous recombination frequency showed that the number of recombination events is reduced in mms21-1 mutant. Conclusively, we provided evidence that AtMMS21 plays an important role in homologous recombination for DNA damage repair.     

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.     

The DNA damage response: sensing and signaling

The protein kinases ATM and ATR are central components of the checkpoint mechanisms that signal the presence of damaged DNA and stalled replication forks. Recent studies have provided important new insights into how these kinases work together with their regulatory subunits, DNA repair proteins and adaptor proteins to sense abnormal DNA structures and implement the appropriate DNA damage response. These advances have provided a more detailed understanding of the interface between damaged DNA and the checkpoint sensor proteins.     

DNA repair, damage signaling and carcinogenesis

The First joint meeting of the German DGDR (German Society for Research on DNA Repair) and the French SFTG (French Society of Genotoxicology) on DNA Repair was held in Toulouse, France, from September 15 to 19, 2007. It was organized by Lisa Wiesmüller and Bernard Salles together with the scientific committee consisting of Gilbert de Murcia, Jean-Marc Egly, Frank Grosse, Karl-Peter Hopfner, Georges Iliakis, Bernd Kaina, Markus Löbrich, Bernard Lopez, Daniel Marzin and Alain Sarasin. This report summarizes information presented by the speakers (invited lectures and oral communications) during the seven plenary sessions, which include (1) excision repair, (2) DNA repair and carcinogenesis, (3) double-strand break repair, (4) replication in repair and lesion bypass, (5) cellular responses to genotoxic stress, (6) DNA repair machinery within the chromatin context and (7) genotoxicology and testing. A total of 23 plenary lectures, 32 oral communications and 66 posters were presented in this rather intense 4 days meeting, which stimulated extensive discussions and highly interdisciplinary scientific exchanges among the ∼250 participants.     

MAD2 interacts with DNA repair proteins and negatively regulates DNA damage repair

MAD2 (mitotic arrest deficient 2) is a key regulator of mitosis. Recently, it had been suggested that MAD2-induced mitotic arrest mediates DNA damage response and that upregulation of MAD2 confers sensitivity to DNA-damaging anticancer drug-induced apoptosis. In this study, we report a potential novel role of MAD2 in mediating DNA nucleotide excision repair through physical interactions with two DNA repair proteins, XPD (xeroderma pigmentosum complementation group D) and ERCC1. First, overexpression of MAD2 resulted in decreased nuclear accumulation of XPD, a crucial step in the initiation of DNA repair. Second, immunoprecipitation experiments showed that MAD2 was able to bind to XPD, which led to competitive suppression of binding activity between XPD and XPA, resulting in the prevention of physical interactions between DNA repair proteins. Third, unlike its role in mitosis, the N-terminus domain seemed to be more important in the binding activity between MAD2 and XPD. Fourth, phosphorylation of H2AX, a process that is important for recruitment of DNA repair factors to DNA double-strand breaks, was suppressed in MAD2-overexpressing cells in response to DNA damage. These results suggest a negative role of MAD2 in DNA damage response, which may be accounted for its previously reported role in promoting sensitivity to DNA-damaging agents in cancer cells. However, the interaction between MAD2 and ERCC1 did not show any effect on the binding activity between ERCC1 and XPA in the presence or absence of DNA damage. Our results suggest a novel function of MAD2 by interfering with DNA repair proteins.     

DNA repair,recombination, and damage signaling

DNA must be accurately copied and propagated from one cell division to the next, and from one generation to the next. To ensure the faithful transmission of the genome, a plethora of distinct as well as overlapping DNA repair and recombination pathways have evolved. These pathways repair a large variety of lesions, including alterations to single nucleotides and DNA single and double-strand breaks, that are generated as a consequence of normal cellular function or by external DNA damaging agents. In addition to the proteins that mediate DNA repair, checkpoint pathways have also evolved to monitor the genome and coordinate the action of various repair pathways. Checkpoints facilitate repair by mediating a transient cell cycle arrest, or through initiation of cell suicide if DNA damage has overwhelmed repair capacity. In this chapter, we describe the attributes of Caenorhabditis elegans that facilitate analyses of DNA repair, recombination, and checkpoint signaling in the context of a whole animal. We review the current knowledge of C. elegans DNA repair, recombination, and DNA damage response pathways, and their role during development, growth, and in the germ line. We also discuss how the analysis of mutational signatures in C. elegans is helping to inform cancer mutational signatures in humans.     

The DNA damage response signaling cascade regulates proliferation of the phytopathogenic fungus Ustilago maydis in planta

In the phytopathogenic fungus Ustilago maydis, the dikaryotic state dominates the period of growth occurring during the infectious phase. Dikaryons are cells in which two nuclei, one from each parent cell, share a single cytoplasm for a period of time without undergoing nuclear fusion. In fungal cells, maintenance of the dikaryotic state requires an intricate cell division process that often involves the formation of a structure known as the clamp connection as well as the sorting of one of the nuclei to this structure to ensure that each daughter dikaryon inherits a balance of each parental genome. Here, we describe an atypical role of the DNA damage checkpoint kinases Chk1 and Atr1 during pathogenic growth of U. maydis. We found that Chk1 and Atr1 collaborate to control cell cycle arrest during the induction of the virulence program in U. maydis and that Chk1 and Atr1 work together to control the dikaryon formation. These findings uncover a link between a widely conserved signaling cascade and the virulence program in a phytopathogen. We propose a model in which adjustment of the cell cycle by the Atr1-Chk1 axis controls fidelity in dikaryon formation. Therefore, Chk1 and Atr1 emerge as critical cell type regulators in addition to their roles in the DNA damage response.     

MCL-1 localizes to sites of DNA damage and regulates DNA damage response

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1^-/- MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G1/S checkpoint response, was not significantly different. In addition, appearance of g-H2AX was delayed in the Mcl-1^-/- MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with g-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide over several days, which mimics the clinical situation of etoposide use, resulted in many more chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.     

MCL-1 localizes to sites of DNA damage and regulates DNA damage response

MCL-1, a pro-survival member of the BCL-2 family, was previously shown to have functions in ATR-dependent Chk1 phosphorylation following DNA damage. To further delineate these functions, we explored possible differences in DNA damage response caused by lack of MCL-1 in mouse embryo fibroblasts (MEFs). As expected, Mcl-1^-/- MEFs had delayed Chk1 phosphorylation following etoposide treatment, compared to wild type MEFs. However, their response to hydroxyurea, which causes a G₁/S checkpoint response, was not significantly different. In addition, appearance of γ-H2AX was delayed in the Mcl-1^-/- MEFs treated with etoposide. We next investigated whether MCL-1 is present, together with other DNA damage response proteins, at the sites of DNA damage. Immunoprecipitation of etoposide-treated extracts with anti-MCL-1 antibody showed association of MCL-1 with γ-H2AX as well as NBS1. Immunofluorescent staining for MCL-1 further showed increased co-staining of MCL-1 and NBS1 following DNA damage. By using a system that creates DNA double strand breaks at specific sites in the genome, we demonstrated that MCL-1 is recruited directly adjacent to the sites of damage. Finally, in a direct demonstration of the importance of MCL-1 in allowing proper repair of DNA damage, we found that treatment for two brief exposures to etoposide, followed by periods of recovery, which mimics the clinical situation of etoposide use, resulted in greater accumulation of chromosomal abnormalities in the MEFs that lacked MCL-1. Together, these data indicate an important role for MCL-1 in coordinating DNA damage mediated checkpoint response, and have broad implications for the importance of MCL-1 in maintenance of genome integrity.Key words: protein complex, DNA repair, checkpoint, G₂/M, chromosomes     

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate–dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21^Cip1 accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.     

UV damage and repair in the domain of the human c-myc oncogene

We have used a modification of the Southern hybridization method to analyze the removal of UV-induced pyrimidine cyclobutane dimers from the domain of the c-myc oncogene. The study was performed in human COLO320HSR cells, which exhibit a 30- to 40-fold amplification of c-myc that is maintained in a marker chromosome as a homogeneously staining region. Intron 2 and the region upstream from the gene showed better dimer removal than intron 1 or the region downstream from the c-myc gene. Regions showing less repair coincide with regions that are hotspots for mutations and chromosome translocations. Therefore, it is proposed that the inefficiency of DNA repair may play an important role in the origin of c-myc rearrangements.     

Acetylation regulates DNA repair mechanisms in human cells

The p300-mediated acetylation of enzymes involved in DNA repair and replication has been previously shown to stimulate or inhibit their activities in reconstituted systems. To explore the role of acetylation on DNA repair in cells we constructed plasmid substrates carrying inactivating damages in the EGFP reporter gene, which should be repaired in cells through DNA mismatch repair (MMR) or base excision repair (BER) mechanisms. We analyzed efficiency of repair within these plasmid substrates in cells exposed to deacetylase and acetyltransferase inhibitors, and also in cells deficient in p300 acetyltransferase. Our results indicate that protein acetylation improves DNA mismatch repair in MMR-proficient HeLa cells and also in MMR-deficient HCT116 cells. Moreover, results suggest that stimulated repair of mismatches in MMR-deficient HCT116 cells is done though a strand-displacement synthesis mechanism described previously for Okazaki fragments maturation and also for the EXOI-independent pathway of MMR. Loss of p300 reduced repair of mismatches in MMR-deficient cells, but did not have evident effects on BER mechanisms, including the long patch BER pathway. Hypoacetylation of the cells in the presence of acetyltransferase inhibitor, garcinol generally reduced efficiency of BER of 8-oxoG damage, indicating that some steps in the pathway are stimulated by acetylation.     

USP7S-dependent inactivation of Mule regulates DNA damage signalling and repair

The E3 ubiquitin ligase Mule/ARF-BP1 plays an important role in the cellular DNA damage response by controlling base excision repair and p53 protein levels. However, how the activity of Mule is regulated in response to DNA damage is currently unknown. Here, we report that the Ser18-containing isoform of the USP7 deubiquitylation enzyme (USP7S) controls Mule stability by preventing its self-ubiquitylation and subsequent proteasomal degradation. We find that in response to DNA damage, downregulation of USP7S leads to self-ubiquitylation and proteasomal degradation of Mule, which eventually leads to p53 accumulation. Cells that are unable to downregulate Mule show reduced ability to upregulate p53 levels in response to DNA damage. We also find that, as Mule inactivation is required for stabilization of base excision repair enzymes, the failure of cells to downregulate Mule after DNA damage results in deficient DNA repair. Our data describe a novel mechanism by which Mule is regulated in response to DNA damage and coordinates cellular DNA damage responses and DNA repair.     

ATM signaling and genomic stability in response to DNA damage

DNA double strand breaks represent the most threatening lesion to the integrity of the genome in cells exposed to ionizing radiation and radiomimetic chemicals. Those breaks are recognized, signaled to cell cycle checkpoints and repaired by protein complexes. The product of the gene (ATM) mutated in the human genetic disorder ataxia-telangiectasia (A-T) plays a central role in the recognition and signaling of DNA damage. ATM is one of an ever growing number of proteins which when mutated compromise the stability of the genome and predispose to tumour development. Mechanisms for recognising double strand breaks in DNA, maintaining genome stability and minimizing risk of cancer are discussed.