Sensor Of DNA Double-strand Breaks Research Articles

CtIP Links DNA Double-Strand Break Sensing to Resection

In response to DNA double-strand breaks (DSBs), cells sense the DNA lesions and then activate the protein kinase ATM. Subsequent DSB resection produces RPA-coated ssDNA that is essential for activation of the DNA damage checkpoint and DNA repair by homologous recombination (HR). However, the biochemical mechanism underlying the transition from DSB sensing to resection remains unclear. Using Xenopus egg extracts and human cells, we show that the tumor suppressor protein CtIP plays a critical role in this transition. We find that CtIP translocates to DSBs, a process dependent on the DSB sensor complex Mre11-Rad50-NBS1, the kinase activity of ATM, and a direct DNA-binding motif in CtIP, and then promotes DSB resection. Thus, CtIP facilitates the transition from DSB sensing to processing: it does so by binding to the DNA at DSBs after DSB sensing and ATM activation and then promoting DNA resection, leading to checkpoint activation and HR.

MRE11 complex links RECQ5 helicase to sites of DNA damage

RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.

A Potential Link between Alternative Splicing of theNBS1Gene and DNA Damage/Environmental Stress

NBS1 forms a multimetric complex with MRE11/RAD50, which acts as the sensor of DNA double-strand breaks (DSBs). The mechanisms controlling the expression of NBS1 remain largely unknown. Here we show that NBS1 is transcribed as both a wild-type and an alternatively spliced form exhibiting a premature stop codon in an alternative 50-bp exon in intron 2. Although the wild-type transcript predominates in most tissues, the spliced transcript is abundant in resting peripheral blood mononuclear cells (PBMCs). Levels of the spliced form of NBS1 decreased rapidly after irradiation as levels of the wild-type NBS1 transcript increased, resulting in increased levels of NBS1 protein. Both mitogenic stimulation and methyl methanesulfonate treatment also altered the splicing pattern of NBS1. Resting PBMCs, which predominantly express spliced NBS1, were more susceptible to radiation than mitogen-stimulated cells, which showed predominant expression of the wild-type transcript. Since the alternatively spliced NBS1 gene likely did not produce protein, this alternative splicing seems to be associated with the control of NBS1 protein. Thus alternative splicing of the NBS1 gene may be associated with the regulation of NBS1 in response to DSBs, DNA alkylation damage, and mitogenic response.

PARP1-dependent Kinetics of Recruitment of MRE11 and NBS1 Proteins to Multiple DNA Damage Sites

Poly(ADP-ribose) polymerase 1 (PARP1) is a nuclear enzyme that is rapidly activated by DNA strand breaks and signals the presence of DNA lesions by attaching ADP-ribose units to chromatin-associated proteins. The therapeutic applications of PARP inhibitors in potentiating the killing action of ionizing radiation have been well documented and are attracting increasing interest as a cancer treatment. However, the initial kinetics underlying the recognition of multiple DNA lesions by PARP1 and how inhibition of PARP potentiates the activity of DNA-damaging agents are unknown. Here we report the spatiotemporal dynamics of PARP1 recruitment to DNA damage induced by laser microirradiation in single living cells. We provide direct evidence that PARP1 is able to accumulate at a locally induced DNA double strand break. Most importantly, we observed that the rapid accumulation of MRE11 and NBS1 at sites of DNA damage requires PARP1. By determining the kinetics of protein assembly following DNA damage, our study reveals the cooperation between PARP1 and the double strand break sensors MRE11 and NBS1 in the close vicinity of a DNA lesion. This may explain the sensitivity of cancer cells to PARP inhibitors.

Ctp1 Is a Cell-Cycle-Regulated Protein that Functions with Mre11 Complex to Control Double-Strand Break Repair by Homologous Recombination

The Mre11-Rad50-Nbs1 (MRN) complex is a primary sensor of DNA double-strand breaks (DSBs). Upon recruitment to DSBs, it plays a critical role in catalyzing 5' --> 3' single-strand resection that is required for repair by homologous recombination (HR). Unknown mechanisms repress HR in G1 phase of the cell cycle during which nonhomologous end-joining (NHEJ) is the favored mode of DSB repair. Here we describe fission yeast Ctp1, so-named because it shares conserved domains with the mammalian tumor suppressor CtIP. Ctp1 is recruited to DSBs where it is essential for repair by HR. Ctp1 is required for efficient formation of RPA-coated single-strand DNA adjacent to DSBs, indicating that it functions with the MRN complex in 5' --> 3' resection. Transcription of ctp1(+) is periodic during the cell cycle, with the onset of its expression coinciding with the start of DNA replication. These data suggest that regulation of Ctp1 underlies cell-cycle control of HR.

A novel function of DNA repair molecule Nbs1 in terminal differentiation of the lens fibre cells and cataractogenesis

The Nbs1 protein, hypomorphic mutant in Nijmegen breakage syndrome (NBS), is a component of the Mre11/Rad50/Nbs1 (M/R/N) complex that acts as a DNA double-strand break sensor and functions in cell cycle checkpoint in response to DNA damage and DNA repair. Here we report that targeted disruption of murine NBS1 gene ( Nbn) in the lens alters the M/R/N complex nuclear localization and results in microphthalmia in mice due to reduced proliferation of the lens epithelial cells. Unexpectedly, all Nbn-deficient lenses develop cataracts at an early age due to altered lens fibre cell differentiation, including disruption of normal lens epithelial and fibre cell architecture and incomplete denucleation of fibre cells, and these changes are independent of the p53 pathway. In addition, Nbn-deficient lenses show dysregulated transcription of various crystallins. Thus, this study implicates a novel function of Nbs1 in terminal differentiation of the lens fibre cells and in cataractogenesis.

Checking Cancer Development

Bartkova et al. and Gorgoulis et al. have uncovered evidence suggesting that, early in the process of cancer development, the DNA damage response (DDR)--which elicits cell death or cell senescence--acts as a barrier to uncontrolled cell proliferation. This could create a selection pressure to inactivate genes involved in the DDR, such as p53, leading to increased genomic instability and tumor progression. Bartkova et al. used immunohistochemistry to show that two components of the DDR, Chk2 and ataxia telangiectasia mutated (ATM), were activated and that the ATM substrates p53 and histone H2AX were phosphorylated in early stages of bladder cancer development. Phosphorylation of Chk2 and ATM was apparent before the development of pronounced genomic instability or the appearance of mutations in p53 and other genes involved in DDR. Chk2 and ATM were activated in premalignant lesions of the breast, colon, and lung but not in normal proliferating cells or inflammatory tissues. Overexpression of oncogenes that promote S phase entry in U-2-OS-derived cells led to DDR activation, suggesting that deregulated DNA replication elicited the response. Consistent with this notion, various premalignant lesions showed abnormalities associated with deregulated entry into S phase, allelic imbalances at genomic sequences that are difficult to replicate, and a functional DDR. Gorgoulis et al. found that premalignant human lung lesions expressed wild-type p53 and showed evidence of the DDR [focal nuclear localization of p53 binding protein 1 (53BP1, a sensor of DNA double-strand breaks), H2AX phosphorylation, Chk2 phosphorylation, enhanced p53 abundance, and apoptotic cells]. Normal tissue did not exhibit a DDR, whereas non-small cell lung carcinomas showed 53BP1 nuclear foci and H2AX and Chk2 phosphorylation with suppression of apoptosis, generally associated with p53 mutation or decreased p53 abundance. Similarly, the DDR was apparent in premalignant skin lesions from patients with malignant melanoma and in a model of hyperplasia in which human skin grafted onto immunodeficient mice was stimulated with growth factors. Again, preneoplastic lesions showed allelic imbalances at genomic sequences prone to DNA double-strand breaks, and the hyperplastic grafts developed allelic imbalances at these sites within a few weeks. Thus, like Bartkova et al. , Gorgoulis et al. concluded that the DDR, driven by replicative stress, is an early anticancer response. Venkitaraman discusses both articles and provides context for this research. J. Bartkova, Z. Hořejši, K. Koed, A. Krämer, F. Tort, K. Zieger, P. Guldberg, M. Sehested, J. M. Nesland, C. Lukas, T. Ørntoft, J. Lukas, J. Bartek, DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature 434 , 864-870 (2005). [PubMed] V. G. Gorgoulis, L.-V. Vassiliou, P. Karakaidos, P. Zacharatos, A. Kotsinas, T. Liloglou, M. Venere, R. A. DiTullio Jr., N. G. Kastrinakis, B. Levy, D. Kletsas, A. Yoneta, M. Herlyn, C. Kittas, T. D. Halazonetis, Activation of the DNA damage checkpoint and genomic instability in human precancerous lesions. Nature 434 , 907-913 (2005). [PubMed] A. R. Venkitaraman, Aborting the birth of cancer. Nature 434 , 829-830 (2005). [PubMed]

Identification and functional consequences of a novel MRE11 mutation affecting 10 Saudi Arabian patients with the ataxia telangiectasia-like disorder

Ten new patients with ataxia telangiectasia-like disorder (ATLD) from three unrelated Saudi Arabian families have been identified aged 5-37 representing the largest cohort of ATLD patients ever identified. They presented with an early-onset, slowly progressive, ataxia plus ocular apraxia phenotype with an absence of tumor development, even in the oldest patient. Extra-neurological features such as telangiectasia, raised alpha-fetoprotein and reduced immunoglobulin levels were absent. No translocations were found in the two investigated patients, and the presence of microcephaly was noted in four out of eight ascertained patients. All patients are homozygous for a novel missense mutation (630G-->C, W210C) of the MRE11 gene. The cellular consequences of this amino acid change, localized in the nuclease domain of the Mre11 protein, have been determined in fibroblast cultures established from two individuals. They showed high constitutive levels of Mre11 and Rad50 proteins compared with cells from normal individuals but a very low level of the Nbs1 protein. After exposure to ionizing radiation, a dose-dependent defect in ataxia telangiectasia mutated (ATM)-serine 1981, p53-serine 15 and Chk2 phosphorylation, and p53 stabilization were noted, together with a failure to form Mre11 foci and enhanced radiation sensitivity. Formation of gammaH2AX foci was similar to that seen in normal fibroblasts under the experimental conditions examined. These results emphasize the importance of functional interactions among the three proteins of the Mre11-Rad50-Nbs1 complex and lend support to a role of this complex as a sensor of DNA double-strand breaks, acting upstream of ATM.

Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks

The mechanisms by which eukaryotic cells sense DNA double-strand breaks (DSBs) in order to initiate checkpoint responses are poorly understood. 53BP1 is a conserved checkpoint protein with properties of a DNA DSB sensor. Here, we solved the structure of the domain of 53BP1 that recruits it to sites of DSBs. This domain consists of two tandem tudor folds with a deep pocket at their interface formed by residues conserved in the budding yeast Rad9 and fission yeast Rhp9/Crb2 orthologues. In vitro, the 53BP1 tandem tudor domain bound histone H3 methylated on Lys 79 using residues that form the walls of the pocket; these residues were also required for recruitment of 53BP1 to DSBs. Suppression of DOT1L, the enzyme that methylates Lys 79 of histone H3, also inhibited recruitment of 53BP1 to DSBs. Because methylation of histone H3 Lys 79 was unaltered in response to DNA damage, we propose that 53BP1 senses DSBs indirectly through changes in higher-order chromatin structure that expose the 53BP1 binding site.