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Appropriate repair of DNA lesions and the inhibition of DNA repair activities at telomeres are crucial to prevent genomic instability. By fuelling the generation of genetic alterations and by compromising cell viability, genomic instability is a driving force in cancer and ageing (1,2).Herewe identifyM AD2L2(also known asMAD2B or REV7) through functional genetic screening as a novel factor controlling DNA repair activities at mammalian telomeres. We show that MAD2L2 accumulates at uncapped telomeres and promotes non-homologous end-joining (NHEJ)-mediated fusion of deprotected chromosome ends and genomic instability. MAD2L2 depletion causes elongated 3' telomeric overhangs, indicating that MAD2L2 inhibits 5' end resection. End resection blocks NHEJ while committing to homology-directed repair, and is under the control of 53BP1, RIF1 and PTIP (3). Consistent with MAD2L2 promoting NHEJ-mediated telomere fusion by inhibiting 5' end resection, knockdown of the nucleases CTIP or EXO1 partially restores telomere-driven genomic instability in MAD2L2-depleted cells. Control of DNA repair by MAD2L2 is not limited to telomeres. MAD2L2 also accumulates and inhibits end resection at irradiation-induced DNA double-strand breaks and promotes end-joining of DNA double-strand breaks in several settings, including during immunoglobulin class switch recombination. These activities of MAD2L2 depend on ATM kinase activity, RNF8, RNF168,53BP1 and RIF1, but not on PTIP, REV1 and REV3, the latter two acting with MAD2L2 in translesion synthesis (4). Together, our data establish MAD2L2 as a crucial contributor to the control of DNA repair activity by 53BP1 that promotes NHEJ by inhibiting 5' end resection downstream of RIF1., As the processes underlying telomere-driven genomic instability are not completely understood, we performed a functional genetic screen to identify telomere-induced genomic instability regulators (TIGIRs). The TIGIR screen relies on well-controlled [...]

Homologous recombination is a mechanism for DNA repair that enables the exchange of genetic information between DNA molecules. Structural analysis reveals how the protein RecA orchestrates this process. Structural data reveal how RecA protein orchestrates recombination., Author(s): Upasana Roy, Eric C. Greene Author Affiliations: Demystifying the D-loop during DNA recombination Our chromosomes are subjected to a constant barrage of DNA-damaging agents that can lead to the [...]

It has long been assumed that p53 suppresses tumor development through induction of apoptosis, possibly with contributions by cell cycle arrest and cell senescence.sup.1,2. However, combined deficiency in these three processes does not result in spontaneous tumor formation as observed upon loss of p53, suggesting the existence of additional mechanisms that are critical mediators of p53-dependent tumor suppression function.sup.3-5. To define such mechanisms, we performed in vivo shRNA screens targeting p53-regulated genes in sensitized genetic backgrounds. We found that knockdown of Zmat3, Ctsf and Cav1, promoted lymphoma/leukemia development only when PUMA and p21, the critical effectors of p53-driven apoptosis, cell cycle arrest and senescence, were also absent. Notably, loss of the DNA repair gene Mlh1 caused lymphoma in a wild-type background, and its enforced expression was able to delay tumor development driven by loss of p53. Further examination of direct p53 target genes implicated in DNA repair showed that knockdown of Mlh1, Msh2, Rnf144b, Cav1 and Ddit4 accelerated MYC-driven lymphoma development to a similar extent as knockdown of p53. Collectively, these findings demonstrate that extensive functional overlap of several p53-regulated processes safeguards against cancer and that coordination of DNA repair appears to be an important process by which p53 suppresses tumor development. In vivo shRNA screens in sensitized genetic backgrounds identify p53-activated target genes involved in DNA repair that enable its tumor suppressor function., Author(s): Ana Janic [sup.1] [sup.2] , Liz J. Valente [sup.1] [sup.2] [sup.3] , Matthew J. Wakefield [sup.1] [sup.4] , Leon Di Stefano [sup.1] , Liz Milla [sup.1] [sup.2] , Stephen [...]

Isoform B of the chromatin-binding protein Brd4 acts to suppress DNA damage response signalling. Brd4 modulates response to DNA damage The detection and repair of damaged DNA is vital for cell survival and the maintenance of an intact genome, but aberrant DNA damage signalling can be a cause of cancer. Here Michael Yaffe and colleagues examine the role of chromatin structure change in the activation of the DNA damage response (DDR). They find that the B isoform of the chromatin binding protein, Brd4, acts to suppress DDR signalling. As it is bound to acetylated histones, this Brd4 isoform recruits the condensin II chromatin remodelling complex, which prevents relaxation of the chromatin necessary for binding by factors that recognize and repair DNA damage. In this way, this Brd4 isoform regulates the strength of the DDR through mechanisms distinct from known transcriptional interactions with the P-TEFb transcriptional complex. DNA damage activates a signalling network that blocks cell-cycle progression, recruits DNA repair factors and/or triggers senescence or programmed cell death.sup.1. Alterations in chromatin structure are implicated in the initiation and propagation of the DNA damage response.sup.2. Here we further investigate the role of chromatin structure in the DNA damage response by monitoring ionizing-radiation-induced signalling and response events with a high-content multiplex RNA-mediated interference screen of chromatin-modifying and -interacting genes. We discover that an isoform of Brd4, a bromodomain and extra-terminal (BET) family member, functions as an endogenous inhibitor of DNA damage response signalling by recruiting the condensin II chromatin remodelling complex to acetylated histones through bromodomain interactions. Loss of this isoform results in relaxed chromatin structure, rapid cell-cycle checkpoint recovery and enhanced survival after irradiation, whereas functional gain of this isoform compacted chromatin, attenuated DNA damage response signalling and enhanced radiation-induced lethality. These data implicate Brd4, previously known for its role in transcriptional control, as an insulator of chromatin that can modulate the signalling response to DNA damage., Author(s): Scott R. Floyd [sup.1] [sup.2] , Michael E. Pacold [sup.1] [sup.3] [sup.4] , Qiuying Huang [sup.1] , Scott M. Clarke [sup.1] , Fred C. Lam [sup.1] , Ian G. [...]

A purpose-built high-resolution array with 93.75% breakpoint accuracy was used to map 4000 patients with diverse diagnoses and identify eight probands harboring microdeletions at TP53 (17p13.1), a DNA copy-number variations (CNVs) which might cause childhood-onset developmental delay (DD) and cancer. The results revealed a possible association of 17p13.1 CNVs with distinct phenotypes depending on the position of the breakpoint with respect to TP53 and called for further studies to determine if other loci in the genome also give rise to phentotypically distinct disorder.

Mononuclear iron-containing oxygenases conduct a diverse variety of oxidation functions in biology (1,2), including the oxidative demethylation of methylated nucleic acids and histones (3,4). Escherichia coli AlkB is the first such enzyme that was discovered to repair methylated nucleic acids (5,6), which are otherwise cytotoxic and/or mutagenic. AlkB human homologues are known to play pivotal roles in various processes (7-11). Here we present structural characterization of oxidation intermediates for these demethylases. Using a chemical cross-linking strategy (12,13), complexes of AlkB-double stranded DNA (dsDNA) containing 1,[N.sup.6]-etheno adenine (ζA), [N.sup.3]-methyl thymine (3-meT) and [N.sup.3]-methyl cytosine (3-meC) are stabilized and crystallized, respectively. Exposing these crystals, grown under anaerobic conditions containing iron(II) and α-ketoglutarate (αKG), to dioxygen initiates oxidation in crystallo. Glycol (from ζA) and hemiaminal (from 3-meT) intermediates are captured; a zwitterionic intermediate (from 3-meC) is also proposed, based on crystallographic observations and computational analysis. The observation of these unprecedented intermediates provides direct support for the oxidative demethylation mechanism for these demethylases. This study also depicts a general mechanistic view of how a methyl group is oxidatively removed from different biological substrates., E. coli AlkB is a prototype of iron-containing dioxygenases that catalyse oxidative demethylation of nucleic acids and histones (3,4). Its human homologue ABH1 (also known as ALKBH1) demethylates 3-meC in [...]

Nucleotide excision repair is a constitutive stress response that eliminates DNA lesions induced by multiple genotoxic agents. Unlike the immune system, which generates billions of immunoglobulins and T cell receptors for antigen recognition, the nucleotide excision repair complex uses only a few generic factors to detect an astounding diversity of DNA modifications. New data favor an unexpected strategy whereby damage recognition is initiated by the detection of abnormal oscillations in the undamaged strand opposite to DNA lesions. Another core subunit recognizes the increased susceptibility of DNA to be kinked at injured sites. We suggest that early nucleotide excision repair factors gain substrate versatility by avoiding direct contacts with modified residues and exploiting instead the altered dynamics of damaged DNA duplexes.

Global nucleotide excision repair is greatly attenuated in terminally differentiated mammalian cells. We observed this phenomenon in human neurons and in macrophages, noting that the transcription-coupled repair pathway remains functional and that there is no significant reduction in levels of excision repair enzymes. We have discovered that ubiquitin-activating enzyme E1 complements the repair deficiency in macrophage extracts, and although there is no reduction in the concentration of E1 upon differentiation, our results indicate a reduction in phosphorylation of El. In preliminary studies, we have identified the basal transcription factor TFIIH as the potential target for ubiquitination. We suggest that this unusual type of regulation at the level of the E1 enzyme is likely to affect numerous cellular processes and may represent a strategy to coordinate multiple phenotypic changes upon differentiation by using E1 as a 'master switch.' DNA repair | TFIIH | ubiquitination | monocytes | differentiated

Mutation is a central biological process whose rates and spectra are influenced by a variety of complex and interacting forces. Although DNA repair pathways are generally known to play key roles in maintaining genetic stability, much remains to be understood about the relative roles of different pathways in preventing the accumulation of mutations and the extent of heterogeneity in pathway-specific repair efficiencies across different genomic regions. In this study we examine mutation processes in base excision repair-deficient (nth-1) and nucleotide excision repair-deficient (xpa-1) Caenorhabditis elegans mutation-accunmlation (MA) lines across 24 regions of the genome and compare our observations to previous data from mismatch repairdeficient (msh-2 and msh-6) and wild-type (N2) MA lines. Drastic variation in both average and locus-specific mutation rates, ranging two orders of magnitude for the latter, was detected among the four sets of repairdeficient MA lines. Our work provides critical insights into the relative roles of three DNA repair pathways in preventing C. elegans mutation accumulation and provides evidence for the presence of pathway-specific DNA repair territories in the C. elegans genome.

Staphylococcus aureus is an important pathogen, causing a wide range of infections including sepsis, wound infections, pneumonia, and catheter-related infections. In several pathogens ClpP proteases were identified by in vivo expression technologies to be important for virulence. Clp proteolytic complexes are responsible for adaptation to multiple stresses by degrading accumulated and misfolded proteins. In this report clpP, encoding the proteolytic subunit of the ATP-dependent Clp protease, was deleted, and gene expression of [DELTA]clpP was determined by global transcriptional analysis using DNA-microarray technology. The transcriptional profile reveals a strong regulatory impact of ClpP on the expression of genes encoding proteins that are involved in the pathogenicity of S. aureus and adaptation of the pathogen to several stresses. Expression of the agr system and agr-dependent extracellular virulence factors was diminished. Moreover, the loss of clpP leads to a complete transcriptional derepression of genes of the CtsR- and HrcA-controlled heat shock regulon and a partial derepression of genes involved in oxidative stress response, metal homeostasis, and SOS DNA repair controlled by PerR, Fur, MntR, and LexA. The levels of transcription of genes encoding proteins involved in adaptation to anaerobic conditions potentially regulated by an Fnr-like regulator were decreased. Furthermore, the expression of genes whose products are involved in autolysis was deregulated, leading to enhanced autolysis in the mutant. Our results indicate a strong impact of ClpP proteolytic activity on virulence, stress response, and physiology in S. aureus.

Agents targeting topoisomerases are active against a wide range of human tumors. Stabilization of covalent complexes, converting topoisomerases into DNA-damaging agents, is an essential aspect of cell killing by these drugs. A unique aspect of the repair of topoisomerase-mediated DNA damage is the requirement for pathways that can remove protein covalently bound to DNA. Tyrosyl-DNA phosphodiesterase (Tdp1) is an enzyme that removes phosphotyrosyl moieties bound to the 3' end of DNA. Cells lacking Tdp1 are hypersensitive to camptothecin, consistent with a role for Tdp1 in processing 3' phosphotyrosyl protein-DNA covalent complexes. Because Top2p forms a 5' phosphotyrosyl linkage with DNA, previous work predicted that Tdp1p would not be active against lesions involving Top2p. We found that deletion of the TDP1 gene in yeast confers hypersensitivity to Top2 targeting agents. Combining tdp1 mutations with deletions of genes involved in nonhomologous end joining, excision repair, or postreplication repair enhanced sensitivity to Top2 targeting drugs over the level seen with single mutants, suggesting that Tdp1 may function in collaboration with multiple pathways involved in strand break repair. tdp1 mutations can sensitize yeast cells to drugs targeting Top2 even when TOP1 is deleted. Finally, bacterially expressed yeast Tdp1p is able to remove a peptide derived from yTop2 that is covalently bound to DNA by a 5' phosphotyrosyl linkage. Our results show that Tdp1 plays more general roles in DNA repair than repair of Top1 mediated DNA damage, and may participate in repairing many types of base damage to DNA. topoisomerase II | etoposide | RAD2 | repair nuclease | RAD52

Young (4- to 6-month-old) and aged (24- to 28-month-old) mice were exposed to 2-nitropropane (2-NP), a DNA oxidizing agent, and the ability to induce DNA polymerase [beta] ([beta]-pol) and AP endonuclease (APE) was determined. In contrast to the inducibility of these gene products in response to oxidative damage in young mice, aged mice showed a lack of inducibility of [beta]-pol and APE. APE protein level and endonuclease activity were both reduced 40% (p < .01) in response to 2-NP. Accordingly, the accumulation of DNA repair intermediates in response to 2-NP differed with age. Young animals accumulated 3'OH-containing DNA strand breaks, whereas the aged animals did not. A role for p53 in the difference in DNA damage response with age is suggested by the observation that the accumulation of p53 protein in response to DNA damage in young animals was absent in the aged animals. Our results are consistent with a reduced ability to process DNA damage with age.

Reverse transcription of retroviral RNA genomes produce a double-stranded linear cDNA molecule. A host degradation system prevents a majority of the cDNA molecules from completing the obligatory genomic integration necessary for pathogenesis. We demonstrate that the human TFIIH complex proteins XPB (ERCC3) and XPD (ERCC2) play a principal role in the degradation of retroviral cDNA. DNA repair-deficient XPB and XPD mutant cell lines exhibited an increase in transduction efficiency by both HIV- and Moloney murine leukemia virus-based retroviral vectors. Replicating Moloney murine leukemia virus viral production was greater in XPB or XPD mutant cells but not XPA mutant cells. Quantitative PCR showed an increase in total cDNA molecules, integrated provirus, and 2LTR circles in XPB and XPD mutant cells. In the presence of a reverse transcription inhibitor, the HIV cDNA appeared more stable in mutant XPB or XPD cells. These studies implicate the nuclear DNA repair proteins XPB and XPD in a cellular defense against retroviral infection. AIDS | ERCC2 | ERCC3 | HIV | xeroderma pigmentosum

Endogenously formed reactive oxygen species continuously damage cellular constituents including DNA. These challenges, coupled with exogenous exposure to agents that generate reactive oxygen species, are both associated with normal aging processes and linked to cardiovascular disease, cancer, cataract formation, and fatty liver disease. Although not all of these diseases have been definitively shown to originate from mutations in nuclear DNA or mitochondrial DNA, repair of oxidized, saturated, and ring-fragmented bases via the base excision repair pathway is known to be critical for maintaining genomic stability. One enzyme that initiates base excision repair of ring-fragmented purines and some saturated pyrimidines is NEIL1, a mammalian homolog to Escherichia coil endonuclease VIII. To investigate the organismal consequences of a deficiency in NEIL1, a knockout mouse model was created. In the absence of exogenous oxidative stress, neil1 knockout (neil[1.sup.-/-]) and heterozygotic (neil[1.sup.+/-]) mice develop severe obesity, dyslipidemia, and fatty liver disease and also have a tendency to develop hyperinsulinemia. In humans, this combination of clinical manifestations, including hypertension, is known as the metabolic syndrome and is estimated to affect >40 million people in the United States. Additionally, mitochondrial DNA from neil[1.sup.-/-] mice show increased levels of steady-state DNA damage and deletions relative to wild-type controls. These data suggest an important role for NEIL1 in the prevention of the diseases associated with the metabolic syndrome. DNA repair | fatty liver disease | mitochondria | obesity | oxidative stress

DNA ligase D (LigD) is a large polyfunctional protein that participates in a recently discovered pathway of nonhomologous endjoining in bacteria. LigD consists of an ATP-dependent ligase domain fused to a polymerase domain (Pol) and a phosphoesterase module. The Pol activity is remarkable for its dependence on manganese, its ability to perform templated and nontemplated primer extension reactions, and its preference for adding ribonucleotides to blunt DNA ends. Here we report the 1.5-A crystal structure of the Pol domain of Pseudomonas LigD and its complexes with manganese and ATP/dATP substrates, which reveal a minimized polymerase with a two-metal mechanism and a fold similar to that of archaeal DNA primase. Mutational analysis highlights the functionally relevant atomic contacts in the active site. Although distinct nucleoside conformations and contacts for ATP versus dATP are observed in the cocrystals, the functional analysis suggests that the ATP-binding mode is the productive conformation for dNMP and rNMP incorporation. We find that a mutation of Mycobacterium LigD that uniquely ablates the polymerase activity results in increased fidelity of blunt-end doublestrand break repair in vivo by virtue of eliminating nucleotide insertions at the recombination junctions. Thus, LigD Pol is a direct catalyst of mutagenic nonhomologous end-joining in vivo. Our studies underscore a previously uncharacterized role for the primase-like polymerase family in DNA repair. DNA repair | double-strand breaks | mycobacteria | Pseudomonas

Rad51 is the primary eukaryotic recombinase responsible for initiating DNA strand exchange during homologous recombination. Although the subject of intense study for over a decade, many molecular details of the reactions promoted by Rad51 and related recombinases remain unknown. Using total internal reflection fluorescence microscopy, we directly visualized the behavior of individual Rad51 complexes on double-stranded DNA (dsDNA) molecules suspended in an extended configuration above a lipid bilayer. Here we show that complexes of Rad51 can bind to and slide freely along the helical axis of dsDNA. Sliding is bidirectional, does not require ATP hydrolysis, and displays properties consistent with a 1D random walk driven solely by thermal diffusion. The proteins move freely on the DNA for long periods of time; however, sliding terminates and the proteins become immobile upon encountering the free end of a linear dsDNA molecule. This study provides previously uncharacterized insights into the behaviors of human Rad51, which may apply to other members of the RecA-like family of recombinases. DNA repair | homologous recombination | total internal reflection fluorescence microscopy

In the yeast Saccharomyces cerevisiae that lacks lamins, the nuclear pore complex (NPC) has been proposed to serve a role in chromatin organization. Here, using fluorescence microscopy in living cells, we show that nuclear pore proteins of the Nup84 core complex, Nup84p, Nup145Cp, Nup120p, and Nup133p, serve to anchor telomere XI-L at the nuclear periphery. The integrity of this complex is shown to be required for repression of a URA3 gene inserted in the subtelomeric region of this chromosome end. Furthermore, altering the integrity of this complex decreases the efficiency of repair of a DNA double-strand break (DSB) only when it is generated in the subtelomeric region, even though the repair machinery is functional. These effects are specific to the Nup84 complex. Our observations thus confirm and extend the role played by the NPC, through the Nup84 complex, in the functional organization of chromatin. They also indicate that anchoring of telomeres is essential for efficient repair of DSBs occurring therein and is important for preserving genome integrity.

Homology directed repair (HDR) and non-homologous end-joining (NHEJ) are the two mechanisms for repairing DNA double-strand breaks (DSBs). The service of RNA transcripts as templates for homology directed repair (HDR) is discussed.

The RSC and Swi/Snf ATP-dependent chromatin-remodeling complexes play key roles in double-strand break (DSB) repair, specifically by homologous recombination (HR). It is shown that Swi/Snf is required earlier, at or preceding the strand invasion step of HR, while RSC is required following synapsis for completion of the recombinational repair event.

To assess DNA repair activity relative to age, in vitro base excision repair assays were performed using brain and liver nuclear extracts prepared from mice of various ages. An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day-old mice. Brain nuclear extracts prepared from old mice showed a decreased abundance of DNA polymerase-[beta], but the addition of purified protein did not restore base excision repair activity. Abundances of other tested base excision repair proteins did not change relative to age. The conclusion is that, during aging, a decline in DNA repair could contribute to increased levels of DNA damage and mutagenesis.

Frame-shift mutations at microsatellites occur as a time-dependent function of polymerase errors followed by failure of postreplicational mismatch repair. A cell-culture system was developed that allows identification of intermediate mutant cells that carry the mutation on a single DNA strand after the initial DNA polymerase errors. A plasmid was constructed that contained 13 repeats of a poly(dC-dA).poly(dG-dT) oligonucleotide immediately after the translation initiation codon of the enhanced GFP (EGFP) gene, shifting the EGFP gene out of its proper reading frame. The plasmid was introduced into human mismatch repair-deficient (HCT116, hMLH1-mutated) and mismatch repair-proficient (HCT116+chr3, hMLH1 wild type) colorectal cancer cells. After frame-shift mutations occurred that restored the EGFP reading frame, EGFP-expressing cells were detected, and two distinct fluorescent populations, M1 (dim cells) and M2 (bright cells), were identified. M1 cell numbers were stable, whereas M2 cells accumulated over time. In HCT116, single M2 cells gave rise to fluorescent colonies that carried a 2-bp deletion at the [(CA).sub.13] microsatellite, Twenty-eight percent of single M1 cells, however, gave rise to colonies with a mixed fluorescence pattern that carried both [(CA).sup.13] and [(CA).sub.12] microsatellites. It is likely that M1 cells represent intermediate mutants that carry [(CA).sub.13]*[(GT).sub.12] heteroduplexes. Although the mutation rate in HCT116 cell clones (6.2 x [10.sup.-4]) was 30 times higher than in HCT116+chr3 (1.9 x [10.sup.-5]), the proportion of M1 cells in culture did not significantly differ between HCT116 (5.87 x [10.sup.-3]) and HCT116+chr3 (4.13 x [10.sup.-3]), indicating that the generation of intermediate mutants is not affected by mismatch-repair proficiency. mismatch repair | frame-shift mutation | microsatellite instability | mutation rate

Research reveals that oligodeoxyribonucleotides containing multiple adducts, repairable by O(sup)6-alkylguanine-DNA alkyltransferase, are useful as inhibitory 'pseudosubstrates' for protein inactivation. Data indicate that O(sup)6-alkylguanine-DNA alkyltransferase recognizes the polarity of single-stranded DNA and this aids in designing of novel O(sup)6-alkylguanine-DNA alkyltransferase inhibitors incorporating O(sup)6-alkylguanine adducts