Systematic characterization of deubiquitylating enzymes for roles in maintaining genome integrity.

DNA double-strand breaks (DSBs) are perhaps the most toxic of all DNA lesions, with defects in the DNA-damage response to DSBs being associated with various human diseases. Although it is known that DSB repair pathways are tightly regulated by ubiquitylation, we do not yet have a comprehensive understanding of how deubiquitylating enzymes (DUBs) function in DSB responses. Here, by carrying out a multidimensional screening strategy for human DUBs, we identify several with hitherto unknown links to DSB repair, the G2/M DNA-damage checkpoint and genome-integrity maintenance. Phylogenetic analyses reveal functional clustering within certain DUB subgroups, suggesting evolutionally conserved functions and/or related modes of action. Furthermore, we establish that the DUB UCHL5 regulates DSB resection and repair by homologous recombination through protecting its interactor, NFRKB, from degradation. Collectively, our findings extend the list of DUBs promoting the maintenance of genome integrity, and highlight their potential as therapeutic targets for cancer.This is the author's accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/ncb302

Smc5/6 coordinates formation and resolution of joint molecules with chromosome morphology to ensure meiotic divisions

During meiosis, Structural Maintenance of Chromosome (SMC) complexes underpin two fundamental features of meiosis: homologous recombination and chromosome segregation. While meiotic functions of the cohesin and condensin complexes have been delineated, the role of the third SMC complex, Smc5/6, remains enigmatic. Here we identify specific, essential meiotic functions for the Smc5/6 complex in homologous recombination and the regulation of cohesin. We show that Smc5/6 is enriched at centromeres and cohesin-association sites where it regulates sister-chromatid cohesion and the timely removal of cohesin from chromosomal arms, respectively. Smc5/6 also localizes to recombination hotspots, where it promotes normal formation and resolution of a subset of joint-molecule intermediates. In this regard, Smc5/6 functions independently of the major crossover pathway defined by the MutLγ complex. Furthermore, we show that Smc5/6 is required for stable chromosomal localization of the XPF-family endonuclease, Mus81-Mms4Eme1. Our data suggest that the Smc5/6 complex is required for specific recombination and chromosomal processes throughout meiosis and that in its absence, attempts at cell division with unresolved joint molecules and residual cohesin lead to severe recombination-induced meiotic catastroph

Cellular Radiosensitivity: How much better do we understand it?

Purpose: Ionizing radiation exposure gives rise to a variety of lesions in DNA that result in genetic instability and potentially tumorigenesis or cell death. Radiation extends its effects on DNA by direct interaction or by radiolysis of H2O that generates free radicals or aqueous electrons capable of interacting with and causing indirect damage to DNA. While the various lesions arising in DNA after radiation exposure can contribute to the mutagenising effects of this agent, the potentially most damaging lesion is the DNA double strand break (DSB) that contributes to genome instability and/or cell death. Thus in many cases failure to recognise and/or repair this lesion determines the radiosensitivity status of the cell. DNA repair mechanisms including homologous recombination (HR) and non-homologous end-joining (NHEJ) have evolved to protect cells against DNA DSB. Mutations in proteins that constitute these repair pathways are characterised by radiosensitivity and genome instability. Defects in a number of these proteins also give rise to genetic disorders that feature not only genetic instability but also immunodeficiency, cancer predisposition, neurodegeneration and other pathologies. Conclusions: In the past fifty years our understanding of the cellular response to radiation damage has advanced enormously with insight being gained from a wide range of approaches extending from more basic early studies to the sophisticated approaches used today. In this review we discuss our current understanding of the impact of radiation on the cell and the organism gained from the array of past and present studies and attempt to provide an explanation for what it is that determines the response to radiation

Mutation screening of the RNF8, UBC13 and MMS2 genes in Northern Finnish breast cancer families

<p>Abstract</p> <p>Background</p> <p>Currently known susceptibility genes such as <it>BRCA1 </it>and <it>BRCA2 </it>explain less than 25% of familial aggregation of breast cancer, which suggests the involvement of additional susceptibility genes. RNF8, UBC13 and MMS2 are involved in the DNA damage response pathway and play important roles in BRCA1-mediated DNA damage recognition. Based on the evidence that several players in the ubiquitin-mediated BRCA1-dependent DDR seem to contribute to breast cancer predisposition, <it>RNF8, UBC13 </it>and <it>MMS2 </it>were considered plausible candidate genes for susceptibility to breast cancer.</p> <p>Methods</p> <p>The entire coding region and splice junctions of <it>RNF8, UBC13 </it>and <it>MMS2 </it>genes were screened for mutations in affected index cases from 123 Northern Finnish breast cancer families by using conformation sensitive gel electrophoresis, high resolution melting (HRM) analysis and direct sequencing.</p> <p>Results</p> <p>Mutation analysis revealed several changes in <it>RNF8 </it>and <it>UBC13</it>, whereas no aberrations were observed in <it>MMS2</it>. None of the found sequence changes appeared to associate with breast cancer susceptibility.</p> <p>Conclusions</p> <p>The present data suggest that mutations in <it>RNF8, UBC13 </it>and <it>MMS2 </it>genes unlikely make any sizeable contribution to breast cancer predisposition in Northern Finland.</p

A siRNA-Based Screen for Genes Involved in Chromosome End Protection

Telomeres are nucleoprotein complexes which protect the ends of linear chromosomes from detection as DNA damage and provide a sequence buffer against replication-associated shortening. In mammals, telomeres consist of repetitive DNA sequence (TTAGGG) and associated proteins. The telomeric core complex is called shelterin and is comprised of the proteins TRF1, TRF2, POT1, TIN2, TPP1 and RAP1. Excessive telomere shortening or de-protection of telomeres through the loss of shelterin subunits allows the detection of telomeres as DNA damage, which can be visualized as DNA damage protein foci at chromosome ends called TIF (Telomere Dysfunction-Induced Foci). We sought to exploit the TIF phenotype as marker for telomere dysfunction to identify novel genes involved in telomere protection by siRNA-mediated knock-down of a set of 386 candidates. Here we report the establishment, specificity and feasibility of such a screen and the results of the genes tested. Only one of the candidate genes showed a unique TIF phenotype comparable to the suppression of the main shelterin components TRF2 or TRF1 and that gene was identified as a TRF1-like pseudogene. We also identified a weak TIF phenotype for SKIIP (SNW1), a splicing factor and transcriptional co-activator. However, the knock-down of SKIIP also induced a general, not telomere-specific DNA damage response, which complicates conclusions about a telomeric role. In summary, this report is a technical demonstration of the feasibility of a cell-based screen for telomere deprotection with the potential of scaling it to a high-throughput approach

Meiosis in Mice without a Synaptonemal Complex

The synaptonemal complex (SC) promotes fusion of the homologous chromosomes (synapsis) and crossover recombination events during meiosis. The SC displays an extensive structural conservation between species; however, a few organisms lack SC and execute meiotic process in a SC-independent manner. To clarify the SC function in mammals, we have generated a mutant mouse strain (Sycp1−/−Sycp3−/−, here called SC-null) in which all known SC proteins have been displaced from meiotic chromosomes. While transmission electron microscopy failed to identify any remnants of the SC in SC-null spermatocytes, neither formation of the cohesion axes nor attachment of the chromosomes to the nuclear membrane was perturbed. Furthermore, the meiotic chromosomes in SC-null meiocytes achieved pre-synaptic pairing, underwent early homologous recombination events and sustained a residual crossover formation. In contrast, in SC-null meiocytes synapsis and MLH1-MLH3-dependent crossovers maturation were abolished, whereas the structural integrity of chromosomes was drastically impaired. The variable consequences that SC inactivation has on the meiotic process in different organisms, together with the absence of SC in some unrelated species, imply that the SC could have originated independently in different taxonomic groups

The Intrinsic Antiviral Defense to Incoming HSV-1 Genomes Includes Specific DNA Repair Proteins and Is Counteracted by the Viral Protein ICP0

Cellular restriction factors responding to herpesvirus infection include the ND10 components PML, Sp100 and hDaxx. During the initial stages of HSV-1 infection, novel sub-nuclear structures containing these ND10 proteins form in association with incoming viral genomes. We report that several cellular DNA damage response proteins also relocate to sites associated with incoming viral genomes where they contribute to the cellular front line defense. We show that recruitment of DNA repair proteins to these sites is independent of ND10 components, and instead is coordinated by the cellular ubiquitin ligases RNF8 and RNF168. The viral protein ICP0 targets RNF8 and RNF168 for degradation, thereby preventing the deposition of repressive ubiquitin marks and counteracting this repair protein recruitment. This study highlights important parallels between recognition of cellular DNA damage and recognition of viral genomes, and adds RNF8 and RNF168 to the list of factors contributing to the intrinsic antiviral defense against herpesvirus infection