DNA double-strand break repair within heterochromatic regions

DNA DSBs (double-strand breaks) represent a critical lesion for a cell, with misrepair being potentially as harmful as lack of repair. In mammalian cells, DSBs are predominantly repaired by non-homologous end-joining or homologous recombination. The kinetics of repair of DSBs can differ widely, and recent studies have shown that the higher-order chromatin structure can dramatically affect the pathway utilized, the rate of repair and the genetic factors required for repair. Studies of the repair of DSBs arising within heterochromatic DNA regions have provided insight into the constraints that higher-order chromatin structure poses on repair and the processing that is uniquely required for the repair of such DSBs. In the present paper, we provide an overview of our current understanding of the process of heterochromatic DSB repair in mammalian cells and consider the evolutionary conservation of the processes.     

ATM/ATR kinases link the synaptonemal complex and DNA double-strand break repair pathway choice

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The dystonia gene THAP1 controls DNA double-strand break repair choice

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Interplays between ATM/Tel1 and ATR/Mec1 in sensing and signaling DNA double-strand breaks

DNA double-strand breaks (DSBs) are highly hazardous for genome integrity because they have the potential to cause mutations, chromosomal rearrangements and genomic instability. The cellular response to DSBs is orchestrated by signal transduction pathways, known as DNA damage checkpoints, which are conserved from yeasts to humans. These pathways can sense DNA damage and transduce this information to specific cellular targets, which in turn regulate cell cycle transitions and DNA repair. The mammalian protein kinases ATM and ATR, as well as their budding yeast corresponding orthologs Tel1 and Mec1, act as master regulators of the checkpoint response to DSBs. Here, we review the early steps of DSB processing and the role of DNA-end structures in activating ATM/Tel1 and ATR/Mec1 in an orderly and reciprocal manner.     

DNA motif associated with meiotic double-strand break regions in Saccharomyces cerevisiae

Meiotic recombination in yeast is initiated by DNA double-strand breaks (DSBs) that occur at preferred sites, distributed along the chromosomes. These DSB sites undergo changes in chromatin structure early in meiosis, but their common features at the level of DNA sequence have not been defined until now. Alignment of 1 kb sequences flanking six well-mapped DSBs has allowed us to define a flexible sequence motif, the CoHR profile, which predicts the great majority of meiotic DSB locations. The 50 bp profile contains a poly(A) tract in its centre and may have several gaps of unrelated sequences over a total length of up to 250 bp. The major exceptions to the correlation between CoHRs and preferred DSB sites are at telomeric regions, where DSBs do not occur. The CoHR sequence may provide the basis for understanding meiosis-induced chromatin changes that enable DSBs to occur at defined chromosomal sites.     

The cyclin A1-CDK2 complex regulates DNA double-strand break repair

Vertebrates express two A-type cyclins; both associate with and activate the CDK2 protein kinase. Cyclin A1 is required in the male germ line, but its molecular functions are incompletely understood. We observed specific induction of cyclin A1 expression and promoter activity after UV and gamma-irradiation which was mediated by p53. cyclin A1-/- cells showed increased radiosensitivity. To unravel a potential role of cyclin A1 in DNA repair, we performed a yeast triple hybrid screen and identified the Ku70 DNA repair protein as a binding partner and substrate of the cyclin A1-CDK2 complex. DNA double-strand break (DSB) repair was deficient in cyclin A1-/- cells. Further experiments indicated that A-type cyclins activate DNA DSB repair by mechanisms that depend on CDK2 activity and Ku proteins. Both cyclin A1 and cyclin A2 enhanced DSB repair by homologous recombination, but only cyclin A1 significantly activated nonhomologous end joining. DNA DSB repair was specific for A-type cyclins because cyclin E was ineffective. These findings establish a novel function for cyclin A1 and CDK2 in DNA DSB repair following radiation damage.     

ATM controls meiotic DNA double-strand break formation and recombination and affects synaptonemal complex organization in plants

Meiosis is a specialized cell division that gives rise to genetically distinct gametic cells. Meiosis relies on the tightly controlled formation of DNA double-strand breaks (DSBs) and their repair via homologous recombination for correct chromosome segregation. Like all forms of DNA damage, meiotic DSBs are potentially harmful and their formation activates an elaborate response to inhibit excessive DNA break formation and ensure successful repair. Previous studies established the protein kinase ATM as a DSB sensor and meiotic regulator in several organisms. Here we show that Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover (CO) formation and synaptonemal complex (SC) organization, all vital for the successful completion of meiosis. We developed a single-molecule approach to quantify meiotic breaks and determined that ATM is essential to limit the number of meiotic DSBs. Local and genome-wide recombination screens showed that ATM restricts the number of interference-insensitive COs, while super-resolution STED nanoscopy of meiotic chromosomes revealed that the kinase affects chromatin loop size and SC length and width. Our study extends our understanding of how ATM functions during plant meiosis and establishes it as an integral factor of the meiotic program.

Arabidopsis ATM acts at multiple steps during DSB formation and processing, as well as crossover formation and synaptonemal complex organization, all vital for the successful completion of meiosis.     

Functional association of poly(ADP-ribose) polymerase with DNA polymerase alpha-primase complex: a link between DNA strand break detection and DNA replication.

Poly(ADP-ribose) polymerase (PARP) is an element of the DNA damage surveillance network evolved by eukaryotic cells to cope with numerous environmental and endogenous genotoxic agents. PARP has been found to be involved in vivo in both cell proliferation and base excision repair of DNA. In this study the interaction between PARP and the DNA polymerase alpha-primase tetramer has been examined. We provide evidence that in proliferating cells: (i) PARP is physically associated with the catalytic subunit of the DNA polymerase alpha-primase tetramer, an association confirmed by confocal microscopy, demonstrating that both enzymes are co-localized at the nuclear periphery of HeLa cells; (ii) this interaction requires the integrity of the second zinc finger of PARP and is maximal during the S and G2/M phases of the cell cycle; (iii) PARP-deficient cells derived from PARP knock-out mice exhibited reduced DNA polymerase activity, compared with the parental cells, a reduction accentuated following exposure to sublethal doses of methylmethanesulfonate. Altogether, the present results strongly suggest that PARP participates in a DNA damage survey mechanism implying its nick-sensor function as part of the control of replication fork progression when breaks are present in the template.     

Polycomb repressive complex 2 contributes to DNA double-strand break repair

Polycomb protein histone methyltransferase, enhancer of Zeste homolog 2 (EZH2), is frequently overexpressed in human malignancy and is implicated in cancer cell proliferation and invasion. However, it is largely unknown whether EZH2 has a role in modulating the DNA damage response. Here, we show that polycomb repressive complex 2 (PRC2) is recruited to sites of DNA damage. This recruitment is independent of histone 2A variant X (H2AX) and the PI-3-related kinases ATM and DNA-PKcs. We establish that PARP activity is required for retaining PRC2 at sites of DNA damage. Furthermore, depletion of EZH2 in cells decreases the efficiency of DSB repair and increases sensitivity of cells to gamma-irradiation. These data unravel a crucial role of PRC2 in determining cancer cellular sensitivity following DNA damage and suggest that therapeutic targeting of EZH2 activity might serve as a strategy for improving conventional chemotherapy in a given malignancy.     

SMC1 coordinates DNA double-strand break repair pathways

The SMC1/SMC3 heterodimer acts in sister chromatid cohesion, and recent data indicate a function in DNA double-strand break repair (DSBR). Since this role of SMC proteins has remained largely elusive, we explored interactions between SMC1 and the homologous recombination (HR) or non-homologous end-joining (NHEJ) pathways for DSBR in Saccharomyces cerevisiae. Analysis of conditional single- and double mutants of smc1-2 with rad52Δ, rad54Δ, rad50Δ or dnl4Δ illustrates a significant contribution of SMC1 to the overall capacity of cells to repair DSBs. smc1 but not smc2 mutants show increased hypersensitivity of HR mutants to ionizing irradiation and to the DNA crosslinking agent cis-platin. Haploid, but not diploid smc1-2 mutants were severely affected in repairing multiple genomic DNA breaks, suggesting a selective role of SMC1 in sister chromatid recombination. smc1-2 mutants were also 15-fold less efficient and highly error-prone in plasmid end-joining through the NHEJ pathway. Strikingly, inactivation of RAD52 or RAD54 fully rescued efficiency and accuracy of NHEJ in the smc1 background. Therefore, we propose coordination of HR and NHEJ processes by Smc1p through interaction with the RAD52 pathway.     

The ACF1 complex is required for DNA double-strand break repair in human cells

DNA double-strand breaks (DSBs) are repaired via?nonhomologous end-joining (NHEJ) or homologous?recombination (HR), but cellular repair processes remain elusive. We show here that the ATP-dependent chromatin-remodeling factors, ACF1 and SNF2H, accumulate rapidly at DSBs and are required for DSB repair in human cells. If the expression of ACF1 or SNF2H is suppressed, cells become extremely sensitive to X-rays and chemical treatments producing DSBs, and DSBs remain unrepaired. ACF1 interacts directly with KU70 and is required for the accumulation of KU proteins at DSBs. The KU70/80 complex becomes physically more associated with the chromatin-remodeling factors of the CHRAC complex, which includes ACF1, SNF2H, CHRAC15, and CHRAC17, after treatments producing DSBs. Furthermore, the frequency of NHEJ as well as HR induced by DSBs in chromosomal DNA is significantly decreased in cells depleted of either of these factors. Thus, ACF1 and its complexes play important roles in DSBs repair.     

Mechanisms of insertions at a DNA double-strand break

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Role of the silkworm argonaute2 homolog gene in double-strand break repair of extrachromosomal DNA

The argonaute protein family provides central components for RNA interference (RNAi) and related phenomena in a wide variety of organisms. Here, we isolated, from a Bombyx mori cell, a cDNA clone named BmAGO2, which is homologous to Drosophila ARGONAUTE2, the gene encoding a repressive factor for the recombination repair of extrachromosomal double-strand breaks (DSBs). RNAi-mediated silencing of the BmAGO2 sequence markedly increased homologous recombination (HR) repair of DSBs in episomal DNA, but had no effect on that in chromosomes. Moreover, we found that RNAi for BmAGO2 enhanced the integration of linearized DNA into a silkworm chromosome via HR. These results suggested that BmAgo2 protein plays an indispensable role in the repression of extrachromosomal DSB repair.     

ERCC1-XPF endonuclease facilitates DNA double-strand break repair

ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSBs). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma irradiation, and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-) Ku86(-/-) fibroblasts were hypersensitive to gamma irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3' overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that, as in yeast, ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86 independent.     

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair.     

Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p

Eukaryotic cells respond to DNA damage by arresting the cell cycle and modulating gene expression to ensure efficient DNA repair. The human ATR kinase and its homolog in yeast, MEC1, play central roles in transducing the damage signal. To characterize the role of the Mec1 pathway in modulating the cellular response to DNA damage, we used DNA microarrays to observe genomic expression in Saccharomyces cerevisiae responding to two different DNA-damaging agents. We compared the genome-wide expression patterns of wild-type cells and mutants defective in Mec1 signaling, including mec1, dun1, and crt1 mutants, under normal growth conditions and in response to the methylating-agent methylmethane sulfonate (MMS) and ionizing radiation. Here, we present a comparative analysis of wild-type and mutant cells responding to these DNA-damaging agents, and identify specific features of the gene expression responses that are dependent on the Mec1 pathway. Among the hundreds of genes whose expression was affected by Mec1p, one set of genes appears to represent an MEC1-dependent expression signature of DNA damage. Other aspects of the genomic responses were independent of Mec1p, and likely independent of DNA damage, suggesting the pleiotropic effects of MMS and ionizing radiation. The complete data set as well as supplemental materials is available at http://www-genome.stanford.edu/mec1.     

Poly(ADP-ribose) polymerase (PARP-1) is not involved in DNA double-strand break recovery

Background  

The cytotoxicity and the rejoining of DNA double-strand breaks induced by γ-rays, H₂O₂ and neocarzinostatin, were investigated in normal and PARP-1 knockout mouse 3T3 fibroblasts to determine the role of poly(ADP-ribose) polymerase (PARP-1) in DNA double-strand break repair.     

Probing the Mec1ATR Checkpoint Activation Mechanism with Small Peptides

Yeast Mec1, the ortholog of human ATR, is the apical protein kinase that initiates the cell cycle checkpoint in response to DNA damage and replication stress. The basal activity of Mec1 kinase is activated by cell cycle phase-specific activators. Three distinct activators stimulate Mec1 kinase using an intrinsically disordered domain of the protein. These are the Ddc1 subunit of the 9-1-1 checkpoint clamp (ortholog of human and Schizosaccharomyces pombe Rad9), the replication initiator Dpb11 (ortholog of human TopBP1 and S. pombe Cut5), and the multifunctional nuclease/helicase Dna2. Here, we use small peptides to determine the requirements for Mec1 activation. For Ddc1, we identify two essential aromatic amino acids in a hydrophobic environment that when fused together are proficient activators. Using this increased insight, we have been able to identify homologous motifs in S. pombe Rad9 that can activate Mec1. Furthermore, we show that a 9-amino acid Dna2-based peptide is sufficient for Mec1 activation. Studies with mutant activators suggest that binding of an activator to Mec1 is a two-step process, the first step involving the obligatory binding of essential aromatic amino acids to Mec1, followed by an enhancement in binding energy through interactions with neighboring sequences.     

Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism

Rad9 is required for the activation of DNA damage checkpoint pathways in budding yeast. Rad9 is phosphorylated after DNA damage in a Mec1- and Tel1-dependent manner and subsequently interacts with Rad53. This Rad9-Rad53 interaction has been suggested to trigger the activation and phosphorylation of Rad53. Here we show that Mec1 controls the Rad9 accumulation at double-strand breaks (DSBs). Rad9 was phosphorylated after DSB induction and associated with DSBs. However, its phosphorylation and association with DSBs were significantly decreased in cells carrying a mec1Delta or kinase-negative mec1 mutation. Mec1 phosphorylated the S/TQ motifs of Rad9 in vitro, the same motifs that are phosphorylated after DNA damage in vivo. In addition, multiple mutations in the Rad9 S/TQ motifs resulted in its defective association with DSBs. Phosphorylation of Rad9 was partially defective in cells carrying a weak mec1 allele (mec1-81), whereas its association with DSBs occurred efficiently in the mec1-81 mutants, as found in wild-type cells. However, the Rad9-Rad53 interaction after DSB induction was significantly decreased in mec1-81 mutants, as it was in mec1Delta mutants. Deletion mutation in RAD53 did not affect the association of Rad9 with DSBs. Our results suggest that Mec1 promotes association of Rad9 with sites of DNA damage, thereby leading to full phosphorylation of Rad9 and its interaction with Rad53.