Processing Of DNA Double-strand Breaks Research Articles

Differences in the recruitment of DNA repair proteins at subtelomeric and interstitial I-SceI endonuclease-induced DNA double-strand breaks

Telomeres are nucleoprotein structures that are required to protect chromosome ends. Dysfunctional telomeres are recognized as DNA double-strand breaks (DSBs), and elicit the activation of a DNA damage response (DDR). We have previously reported that DSBs near telomeres are poorly repaired, resulting in a high frequency of large deletions and gross chromosome rearrangements (GCRs). Our previous genetic studies have demonstrated that this sensitivity of telomeric regions to DSBs is a result of excessive processing. In the current study, we have further investigated the sensitivity of telomeric regions to DSBs through the analysis of repair proteins associated with DSBs at interstitial and telomeric sites. Following the inducible expression of I-SceI endonuclease, chromatin immunoprecipitation (ChIP) and real-time quantitative PCR were used to compare the recruitment of repair proteins at I-SceI-induced DSBs at interstitial and subtelomeric sites. We observed that proteins that are specifically associated with processing of DSBs during homologous recombination repair, RAD51, BRCA1, and CtIP, are present at a much greater abundance at subtelomeric DSBs. In contrast, Ku70, which is specifically involved in classical nonhomologous end joining, showed no difference at interstitial and subtelomeric DSBs. Importantly, ATM was lower in abundance at subtelomeric DSBs, while ATR was in greater abundance at subtelomeric DSBs, consistent with the accumulation of processed DSBs near telomeres, since processing is accompanied by a transition from ATM to ATR binding. Combined, our results suggest that excessive processing is responsible for the increased frequency of large deletions and GCRs at DSBs near telomeres.

The Lys63-deubiquitylating Enzyme BRCC36 Limits DNA Break Processing and Repair

Multisubunit protein assemblies offer integrated functionalities for efficient cell signal transduction control. One example of such protein assemblies, the BRCA1-A macromolecular complex, couples ubiquitin recognition and metabolism and promotes cellular responses to DNA damage. Specifically, the BRCA1-A complex not only recognizes Lys(63)-linked ubiquitin (K63-Ub) adducts at the damaged chromatin but is endowed with K63-Ub deubiquitylase (DUB) activity. To explore how the BRCA1-A DUB activity contributes to its function at DNA double strand breaks (DSBs), we used RNAi and genome editing approaches to target BRCC36, the protein subunit that confers the BRCA1-A complex its DUB activity. Intriguingly, we found that the K63-Ub DUB activity, although dispensable for maintaining the integrity of the macromolecular protein assembly, is important in enforcing the accumulation of the BRCA1-A complex onto DSBs. Inactivating BRCC36 DUB attenuated BRCA1-A functions at DSBs and led to unrestrained DSB end resection and hyperactive DNA repair. Together, our findings uncover a pivotal role of BRCC36 DUB in limiting DSB processing and repair and illustrate how cells may physically couple ubiquitin recognition and metabolizing activities for fine tuning of DNA repair processes.

EXD2 - a new player joins the DSB resection team

EXD2 - a new player joins the DSB resection team

Dna2 nuclease-helicase structure, mechanism and regulation by Rpa.

The Dna2 nuclease-helicase maintains genomic integrity by processing DNA double-strand breaks, Okazaki fragments and stalled replication forks. Dna2 requires ssDNA ends, and is dependent on the ssDNA-binding protein Rpa, which controls cleavage polarity. Here we present the 2.3 Å structure of intact mouse Dna2 bound to a 15-nucleotide ssDNA. The nuclease active site is embedded in a long, narrow tunnel through which the DNA has to thread. The helicase domain is required for DNA binding but not threading. We also present the structure of a flexibly-tethered Dna2-Rpa interaction that recruits Dna2 to Rpa-coated DNA. We establish that a second Dna2-Rpa interaction is mutually exclusive with Rpa-DNA interactions and mediates the displacement of Rpa from ssDNA. This interaction occurs at the nuclease tunnel entrance and the 5' end of the Rpa-DNA complex. Hence, it only displaces Rpa from the 5' but not 3' end, explaining how Rpa regulates cleavage polarity.

Processing by MRE11 is involved in the sensitivity of subtelomeric regions to DNA double-strand breaks.

The caps on the ends of chromosomes, called telomeres, keep the ends of chromosomes from appearing as DNA double-strand breaks (DSBs) and prevent chromosome fusion. However, subtelomeric regions are sensitive to DSBs, which in normal cells is responsible for ionizing radiation-induced cell senescence and protection against oncogene-induced replication stress, but promotes chromosome instability in cancer cells that lack cell cycle checkpoints. We have previously reported that I-SceI endonuclease-induced DSBs near telomeres in a human cancer cell line are much more likely to generate large deletions and gross chromosome rearrangements (GCRs) than interstitial DSBs, but found no difference in the frequency of I-SceI-induced small deletions at interstitial and subtelomeric DSBs. We now show that inhibition of MRE11 3′–5′ exonuclease activity with Mirin reduces the frequency of large deletions and GCRs at both interstitial and subtelomeric DSBs, but has little effect on the frequency of small deletions. We conclude that large deletions and GCRs are due to excessive processing of DSBs, while most small deletions occur during classical nonhomologous end joining (C-NHEJ). The sensitivity of subtelomeric regions to DSBs is therefore because they are prone to undergo excessive processing, and not because of a deficiency in C-NHEJ in subtelomeric regions.

Alternative end-joining repair pathways are the ultimate backup for abrogated classical non-homologous end-joining and homologous recombination repair: Implications for the formation of chromosome translocations

DNA double strand breaks (DSB) are the most deleterious lesions for the integrity of the genome, as their misrepair can lead to the formation of chromosome translocations. Cells have evolved two main repair pathways to suppress the formation of these genotoxic lesions: homology-dependent, error-free homologous recombination repair (HRR), and potentially error-prone, classical, DNA-PK-dependent non-homologous end-joining (c-NHEJ). The most salient feature of c-NHEJ, speed, will largely suppress chromosome translocation formation, while sequence alterations at the junction remain possible. It is now widely accepted that when c-NHEJ is inactivated, globally or locally, an alternative form of end-joining (alt-EJ) removes DSBs. Alt-EJ operates with speed and fidelity markedly lower than c-NHEJ, causing thus with higher probability chromosome translocations, and generating more extensive sequence alterations at the junction. Our working hypothesis is that alt-EJ operates as a backup to c-NHEJ. Recent results show that alt-EJ can also backup abrogated HRR in G2 phase cells, again at the cost of elevated formation of chromosome translocations. These observations raise alt-EJ to a global rescuing mechanism operating on ends that have lost their chromatin context in ways that compromise processing by HRR or c-NHEJ. While responsible for eliminating from the genome highly cytotoxic DNA ends, alt-EJ provides this function at the price of increased translocation formation. Here, we analyze recent literature on the mechanisms of chromosome translocation formation and propose a functional hierarchy among DSB processing pathways that makes alt-EJ the global backup pathway. We discuss possible ramifications of this model in cellular DSB management and pathway choice, and analyze its implications in radiation carcinogenesis and the design of novel therapeutic approaches.

ARID1A Deficiency Impairs the DNA Damage Checkpoint and Sensitizes Cells to PARP Inhibitors.

ARID1A, SWI/SNF chromatin remodeling complex subunit, is a recently identified tumor suppressor that is mutated in a broad spectrum of human cancers. Thus, it is of fundamental clinical importance to understand its molecular functions and determine whether ARID1A deficiency can be exploited therapeutically. In this article, we report a key function of ARID1A in regulating the DNA damage checkpoint. ARID1A is recruited to DNA double-strand breaks (DSB) via its interaction with the upstream DNA damage checkpoint kinase ATR. At the molecular level, ARID1A facilitates efficient processing of DSB to single-strand ends and sustains DNA damage signaling. Importantly, ARID1A deficiency sensitizes cancer cells to PARP inhibitors in vitro and in vivo, providing a potential therapeutic strategy for patients with ARID1A-mutant tumors. ARID1A has been identified as one of the most frequently mutated genes across human cancers. Our data suggest that clinical utility of PARP inhibitors might be extended beyond patients with BRCA mutations to a larger group of patients with ARID1A-mutant tumors, which may exhibit therapeutic vulnerability to PARP inhibitors.

Inhibition of Homologous Recombination and Promotion of Mutagenic Repair of DNA Double-Strand Breaks Underpins Arabinoside-Nucleoside Analogue Radiosensitization.

In concurrent chemoradiotherapy, drugs are used to sensitize tumors to ionizing radiation. Although a spectrum of indications for simultaneous treatment with drugs and radiation has been defined, the molecular mechanisms underpinning tumor radiosensitization remain incompletely characterized for several such combinations. Here, we investigate the mechanisms of radiosensitization by the arabinoside nucleoside analogue 9-β-D-arabinofuranosyladenine (araA) placing particular emphasis on the repair of DNA double-strand breaks (DSB), and compare the results to those obtained with fludarabine (F-araA) and cytarabine (araC). Postirradiation treatment with araA strongly sensitizes cells to ionizing radiation, but leaves unchanged DSB repair by NHEJ in logarithmically growing cells, in sorted G1 or G2 phase populations, as well as in cells in the plateau phase of growth. Notably, araA strongly inhibits DSB repair by homologous recombination (HRR), as assessed by scoring ionizing radiation-induced RAD51 foci, and in functional assays using integrated reporter constructs. Cells compromised in HRR by RNAi-mediated transient knockdown of RAD51 show markedly reduced radiosensitization after treatment with araA. Remarkably, mutagenic DSB repair compensates for HRR inhibition in araA-treated cells. Compared with araA, F-araA and araC are only modestly radiosensitizing under the conditions examined. We propose that the radiosensitizing potential of nucleoside analogues is linked to their ability to inhibit HRR and concomitantly promote the error-prone processing of DSBs. Our observations pave the way to treatment strategies harnessing the selective inhibitory potential of nucleoside analogues and the development of novel compounds specifically utilizing HRR inhibition as a means of tumor cell radiosensitization.

BRCA1 and BRCA2 protect against oxidative DNA damage converted into double-strand breaks during DNA replication.

BRCA1 and BRCA2 mutation carriers are predisposed to develop breast and ovarian cancers, but the reasons for this tissue specificity are unknown. Breast epithelial cells are known to contain elevated levels of oxidative DNA damage, triggered by hormonally driven growth and its effect on cell metabolism. BRCA1- or BRCA2-deficient cells were found to be more sensitive to oxidative stress, modeled by treatment with patho-physiologic concentrations of hydrogen peroxide. Hydrogen peroxide exposure leads to oxidative DNA damage induced DNA double strand breaks (DSB) in BRCA-deficient cells causing them to accumulate in S-phase. In addition, after hydrogen peroxide treatment, BRCA deficient cells showed impaired Rad51 foci which are dependent on an intact BRCA1-BRCA2 pathway. These DSB resulted in an increase in chromatid-type aberrations, which are characteristic for BRCA1 and BRCA2-deficient cells. The most common result of oxidative DNA damage induced processing of S-phase DSB is an interstitial chromatid deletion, but insertions and exchanges were also seen in BRCA deficient cells. Thus, BRCA1 and BRCA2 are essential for the repair of oxidative DNA damage repair intermediates that persist into S-phase and produce DSB. The implication is that oxidative stress plays a role in the etiology of hereditary breast cancer.

Abstract IA11: New insights into DNA double-strand break responses

Abstract Repair of damaged DNA in cells is complicated by the necessity to repair the damage in the context of complex chromatin structures. Elucidation of molecular mechanisms of DNA repair in intact cells has been challenging. We developed a system that can rapidly induce DNA double strand breaks (DSBs) at defined endogenous sites in mammalian genomes and enables direct assessment of repair and monitoring of protein recruitment, egress, and modification at DSBs in viable cells. The tight regulation of the system also permits assessments of relative kinetics and dependencies of events associated with cellular responses to DNA breakage. Using chromatin immunoprecipitation, we found that nucleosomes are partially disassembled around DSBs during non-homologous end-joining repair in G1-arrested mammalian cells, characterized by a transient loss of the H2A/H2B histone dimer. Nucleolin, a protein with histone chaperone activity, interacts with RAD50 via its RGG domain and is rapidly recruited to DSBs in an MRE11-NBS1-RAD50 complex dependent manner. Down-regulation of nucleolin abrogates the nucleosome disruption, the recruitment of repair factors, and repair of the DSB, demonstrating for the first time the functional importance of nucleosome disruption in DSB repair and identifying a chromatin remodeling protein required for the process. Interestingly, the nucleosome disruption that occurs during DSB repair in cycling cells differs in that both H2A/H2B and H3/H4 histone dimers are removed. This complete nucleosome disruption is also dependent on nucleolin and is required for RPA recruitment to DSBs, a marker of DSB processing that is a requisite for homologous recombination repair. Since inhibition of nucleolin function blunts the DSB repair process, it has become a potential therapeutic target for radiosensitization of tumors. Similarly, small molecule inhibitors of PIKK kinases like ATM and DNA-PK have been developed and are potent radiosensitizers. The potential clinical use of such compounds will be discussed. Citation Format: Michael B. Kastan. New insights into DNA double-strand break responses. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Susceptibility and Cancer Susceptibility Syndromes; Jan 29-Feb 1, 2014; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(23 Suppl):Abstract nr IA11. doi:10.1158/1538-7445.CANSUSC14-IA11

Abstract 1741: Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ)

Abstract IGF-1R is frequently up-regulated in cancers including melanoma, and is implicated in mediating resistance to chemotherapy and targeted agents via regulation of apoptosis. Here, we investigated responses to temozolomide (TMZ), a methylating agent that generates toxic O6-methylguanine (O6meG) adducts, which are removed by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6meG adducts trigger futile cycles of mismatch repair, ultimately leading to formation of replication-associated DSBs. In a first approach to test links between IGF-1R and response to TMZ, we used a panel of 10 human melanoma cell lines in which we characterized MGMT expression, activation and expression of IGF axis components, and growth inhibitory effects of TMZ. We found that TMZ GI50 values correlated with MGMT levels (r=0.79, p=0.009), and in 7 MGMT-proficient cell lines, with phospho-IGF-1R (r=0.81, p=0.038) suggesting a link between TMZ resistance and IGF-1R activation. In a second approach we used IGF-1R tyrosine kinase inhibitors (TKIs) AZ12253801 and linsitinib (OSI-906), which is currently undergoing clinical evaluation, to assess effects of blocking IGF signaling. These IGF-1R TKIs were capable of sensitizing wild-type and mutant BRAF melanoma cells to TMZ; lack of correlation with apoptosis induction suggested that other factors contributed to this effect. IGF-1R inhibition did not influence MGMT expression or activity, was not epistatic with MGMT inhibition, and TMZ-sensitized MGMT-null cells, suggesting that chemo-sensitization was MGMT-independent. We found that IGF-1R TKI sensitized melanoma cells to inhibition of poly(ADP ribose) polymerase (PARP), and influenced processing of TMZ-induced DSBs, with increased RPA focus formation within 24hr of TMZ application, and persistence of RAD51 foci at 72hr. These data suggest functional impairment of homologous recombination (HR)-mediated DSB repair. Compared with simultaneous or prior IGF-1R TKI, TMZ sensitization was greater when IGF-1R TKI followed TMZ, consistent with a model in which IGF-1R inhibitor pre-treatment reduces DSB yield. In mice bearing melanoma xenografts with a clinically relevant (V600E BRAF, wild-type p53) genotype, this sequential (TMZ→linsitinib) combination treatment was tolerable, and linsitinib achieved peak plasma levels comparable to clinical Cmax values. TMZ and linsitinib caused minor inhibition of tumor growth (gradient reduction of 13% and 25% respectively), while the combination caused supra-additive growth delay (72%) that was significantly different from control (p<0.01), TMZ (p<0.01) and linsitinib (p<0.05) groups. In summary, IGF-1R inhibition influences HR repair of TMZ-induced DSBs and sensitizes melanomas to TMZ using treatment schedules relevant to clinical practice. Citation Format: Roger N. Ramcharan, Tamara Aleksic, Shan Gao, Jordan Tanner, Nicholas Darvill, Esther Bridges, Ruth Asher, Amanda J. Watson, Geoffrey P. Margison, Emmanouela Repapi, Ji-Liang Li, Mark R. Middleton, Valentine M. Macaulay. Inhibition of type 1 insulin-like growth factor receptor (IGF-1R) influences processing of replication-associated DNA double-strand breaks (DSBs) and induces schedule-dependent sensitization of human melanoma to temozolomide (TMZ). [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1741. doi:10.1158/1538-7445.AM2014-1741

A quantitative model of the major pathways for radiation-induced DNA double-strand break repair.

We have developed a model approach to simulate the major pathways of DNA double-strand break (DSB) repair in mammalian and human cells. The proposed model shows a possible mechanistic explanation of the basic regularities of DSB processing through the non-homologous end-joining (NHEJ), homologous recombination (HR), single-strand annealing (SSA) and two alternative end-joining pathways. It reconstructs the time-courses of radiation-induced foci specific to particular repair processes including the major intermediate stages. The model is validated for ionizing radiations of a wide range of linear energy transfer (0.2-236 keV/µm) including a relatively broad spectrum of heavy ions. The appropriate set of reaction rate constants was suggested to satisfy the kinetics of DSB rejoining for the considered types of exposure. The simultaneous assessment of several repair pathways allows to describe their possible biological relations in response to irradiation. With the help of the proposed approach, we reproduce several experimental data sets on γ-H2AX foci remaining in different types of cells including those defective in NHEJ, HR, or SSA functions. The results produced confirm the hypothesis suggesting existence of at least two alternative Ku-independent end-joining pathways.

ATP puts the brake on DNA double-strand break repair: a new study shows that ATP switches the Mre11-Rad50-Nbs1 repair factor between signaling and processing of DNA ends.

DNA double-strand breaks (DSBs) are one of the most deleterious forms of DNA damage and can result in cell inviability or chromosomal aberrations. The Mre11-Rad50-Nbs1 (MRN) ATPase-nuclease complex is a central player in the cellular response to DSBs and is implicated in the sensing and nucleolytic processing of DSBs, as well as in DSB signaling by activating the cell cycle checkpoint kinase ATM. ATP binding to Rad50 switches MRN from an open state with exposed Mre11 nuclease sites to a closed state with partially buried nuclease sites. The functional meaning of this switch remained unclear. A new study shows that ATP binding to Rad50 promotes DSB recognition, tethering, and ATM activation, while ATP hydrolysis opens the nuclease active sites to promote processing of DSBs. MRN thus emerges as functional switch that may coordinate the temporal transition from signaling to processing of DSBs.

RECQ helicase RECQL4 participates in non-homologous end joining and interacts with the Ku complex.

RECQL4, a member of the RecQ helicase family, is a multifunctional participant in DNA metabolism. RECQL4 protein participates in several functions both in the nucleus and in the cytoplasm of the cell, and mutations in human RECQL4 are associated with three genetic disorders: Rothmund-Thomson, RAPADILINO and Baller-Gerold syndromes. We previously reported that RECQL4 is recruited to laser-induced DNA double-strand breaks (DSB). Here, we have characterized the functional roles of RECQL4 in the non-homologous end joining (NHEJ) pathway of DSB repair. In an in vitro NHEJ assay that depends on the activity of DNA-dependent protein kinase (DNA-PK), extracts from RECQL4 knockdown cells display reduced end-joining activity on DNA substrates with cohesive and non-cohesive ends. Depletion of RECQL4 also reduced the end joining activity on a GFP reporter plasmid in vivo. Knockdown of RECQL4 increased the sensitivity of cells to γ-irradiation and resulted in accumulation of 53BP1 foci after irradiation, indicating defects in the processing of DSB. We find that RECQL4 interacts with the Ku70/Ku80 heterodimer, part of the DNA-PK complex, via its N-terminal domain. Further, RECQL4 stimulates higher order DNA binding of Ku70/Ku80 to a blunt end DNA substrate. Taken together, these results implicate that RECQL4 participates in the NHEJ pathway of DSB repair via a functional interaction with the Ku70/Ku80 complex. This is the first study to provide both in vitro and in vivo evidence for a role of a RecQ helicase in NHEJ.

Saccharomyces cerevisiae Rif1 cooperates with MRX-Sae2 in promoting DNA-end resection

Diverse roles in DNA metabolism have been envisaged for budding yeast and mammalian Rif1. In particular, yeast Rif1 is involved in telomere homeostasis, while its mammalian counterpart participates in the cellular response to DNA double-strand breaks (DSBs). Here, we show that Saccharomyces cerevisiae Rif1 supports cell survival to DNA lesions in the absence of MRX or Sae2. Furthermore, it contributes to the nucleolytic processing (resection) of DSBs. This Rif1-dependent control of DSB resection becomes important for DSB repair by homologous recombination when resection activities are suboptimal.

Phosphorylation of the Sae2 endonuclease promotes its solubility and activity in DNA repair (735.4)

The Sae2 protein is an endonuclease that is important for DNA double‐strand break processing together with the Mre11/Rad50/Xrs2 (MRX) complex in budding yeast. Sae2 is phosphorylated by cyclin‐dependent kinase, and this modification was shown to be essential for the coordination of DNA end resection with the S and G2 phases of the cell cycle. Here we map all of the modifications on Sae2 isolated from yeast, finding that there are constitutive as well as damage‐induced phosphorylation and acetylation events. Mutagenesis of these sites indicates that a C‐terminal cluster is most essential for the function of Sae2 in DNA damage survival, and reconstitution of Tel1 (ATM) activity on Sae2 with CDK and MRX in vitro shows that CDK phosphorylation primes these Tel1 modifications.From our experience with recombinant Sae2 expressed in E. coli, we knew that Sae2 forms a variety of oligomeric forms and that the smaller forms are more active than the larger forms in DNA binding and endonuclease activity. Here we find that Sae2 also forms oligomeric complexes in yeast, that these convert transiently into smaller units during DNA damage, and that mutation of the CDK and Tel1 phosphorylation sites alters this transition. Similarly we find that a large percentage of Sae2 appears to be insoluble in the absence of DNA damage, but a pool of Sae2 is transiently soluble. From these results we develop a working model of Sae2 regulation in which a potentially toxic, structure‐specific nuclease is held in an inactive state in normal cells but can be quickly released by post‐translational modifications to become active. Rapid degradation (through both the proteasome and through autophagy) then removes the active protein, returning the pool to an inactive state.

Markov mean properties for cell death-related protein classification

The cell death (CD) is a dynamic biological function involved in physiological and pathological processes. Due to the complexity of CD, there is a demand for fast theoretical methods that can help to find new CD molecular targets. The current work presents the first classification model to predict CD-related proteins based on Markov Mean Properties. These protein descriptors have been calculated with the MInD-Prot tool using the topological information of the amino acid contact networks of the 2423 protein chains, five atom physicochemical properties and the protein 3D regions. The Machine Learning algorithms from Weka were used to find the best classification model for CD-related protein chains using all 20 attributes. The most accurate algorithm to solve this problem was K*. After several feature subset methods, the best model found is based on only 11 variables and is characterized by the Area Under the Receiver Operating Characteristic Curve (AUROC) of 0.992 and the true positive rate (TP Rate) of 88.2% (validation set). 7409 protein chains labeled with “unknown function” in the PDB Databank were analyzed with the best model in order to predict the CD-related biological activity. Thus, several proteins have been predicted to have CD-related function in Homo sapiens: 3DRX–involved in virus-host interaction biological process, protein homooligomerization; 4DWF–involved in cell differentiation, chromatin modification, DNA damage response, protein stabilization; 1IUR–involved in ATP binding, chaperone binding; 1J7D–involved in DNA double-strand break processing, histone ubiquitination, nucleotide-binding oligomerization; 1UTU–linked with DNA repair, regulation of transcription; 3EEC–participating to the cellular membrane organization, egress of virus within host cell, class mediator resulting in cell cycle arrest, negative regulation of ubiquitin-protein ligase activity involved in mitotic cell cycle and apoptotic process. Other proteins from bacteria predicted as CD-related are 2G3V - a CAG pathogenicity island protein 13 from Helicobacter pylori, 4G5A - a hypothetical protein in Bacteroides thetaiotaomicron, 1YLK–involved in the nitrogen metabolism of Mycobacterium tuberculosis, and 1XSV - with possible DNA/RNA binding domains. The results demonstrated the possibility to predict CD-related proteins using molecular information encoded into the protein 3D structure. Thus, the current work demonstrated the possibility to predict new molecular targets involved in cell-death processes.

NBN Gene Polymorphisms and Cancer Susceptibility: A Systemic Review.

The relationship between DNA repair failure and cancer is well established as in the case of rare, high penetrant genes in high cancer risk families. Beside this, in the last two decades, several studies have investigated a possible association between low penetrant polymorphic variants in genes devoted to DNA repair pathways and risk for developing cancer. This relationship would be also supported by the observation that DNA repair processes may be modulated by sequence variants in DNA repair genes, leading to susceptibility to environmental carcinogens. In this framework, the aim of this review is to provide the reader with the state of the art on the association between common genetic variants and cancer risk, limiting the attention to single nucleotide polymorphisms (SNPs) of the NBN gene and providing the various odd ratios (ORs). In this respect, the NBN protein, together with MRE11 and RAD50, is part of the MRN complex which is a central player in the very early steps of sensing and processing of DNA double-strand breaks (DSBs), in telomere maintenance, in cell cycle control, and in genomic integrity in general. So far, many papers were devoted to ascertain possible association between common synonymous and non-synonymous NBN gene polymorphisms and increased cancer risk. However, the results still remain inconsistent and inconclusive also in meta-analysis studies for the most investigated E185Q NBN miscoding variant.

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.

Ionizing radiation and genetic risks. XVII. Formation mechanisms underlying naturally occurring DNA deletions in the human genome and their potential relevance for bridging the gap between induced DNA double-strand breaks and deletions in irradiated germ cells

While much is known about radiation-induced DNA double-strand breaks (DSBs) and their repair, the question of how deletions of different sizes arise as a result of the processing of DSBs by the cell's repair systems has not been fully answered. In order to bridge this gap between DSBs and deletions, we critically reviewed published data on mechanisms pertaining to: (a) repair of DNA DSBs (from basic studies in this area); (b) formation of naturally occurring structural variation (SV) - especially of deletions - in the human genome (from genomic studies) and (c) radiation-induced mutations and structural chromosomal aberrations in mammalian somatic cells (from radiation mutagenesis and radiation cytogenetic studies). The specific aim was to assess the relative importance of the postulated mechanisms in generating deletions in the human genome and examine whether empirical data on radiation-induced deletions in mouse germ cells are consistent with predictions of these mechanisms. The mechanisms include (a) NHEJ, a DSB repair process that does not require any homology and which functions in all stages of the cell cycle (and is of particular relevance in G0/G1); (b) MMEJ, also a DSB repair process but which requires microhomology and which presumably functions in all cell cycle stages; (c) NAHR, a recombination-based DSB repair mechanism which operates in prophase I of meiosis in germ cells; (d) MMBIR, a microhomology-mediated, replication-based mechanism which operates in the S phase of the cell cycle, and (e) strand slippage during replication (involved in the origin of small insertions and deletions (INDELs). Our analysis permits the inference that, between them, these five mechanisms can explain nearly all naturally occurring deletions of different sizes identified in the human genome, NAHR and MMBIR being potentially more versatile in this regard. With respect to radiation-induced deletions, the basic studies suggest that those arising as a result of the operation of NHEJ/MMEJ processes, as currently formulated, are expected to be relatively small. However, data on induced mutations in mouse spermatogonial stem cells (irradiation in G0/G1 phase of the cell cycle and DSB repair presumed to be via NHEJ predominantly) show that most are associated with deletions of different sizes, some in the megabase range. There is thus a 'discrepancy' between what the basic studies suggest and the empirical observations in mutagenesis studies. This discrepancy, however, is only an apparent but not a real one. It can be resolved by considering the issue of deletions in the broader context of and in conjunction with the organization of chromatin in chromosomes and nuclear architecture, the conceptual framework for which already exists in studies carried out during the past fifteen years or so. In this paper, we specifically hypothesize that repair of DSBs induced in chromatin loops may offer a basis to explain the induction of deletions of different sizes and suggest an approach to test the hypothesis. We emphasize that the bridging of the gap between induced DSB and resulting deletions of different sizes is critical for current efforts in computational modeling of genetic risks.