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A mutation-promotive role of nucleotide excision repair in cell cycle-arrested cell populations following UV irradiation.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Growing attention is paid to the concept that mutations arising in stationary, non-proliferating cell populations considerably contribute to evolution, aging, and pathogenesis. If such mutations are beneficial to the affected cell, in the sense of allowing a restart of proliferation, they are called adaptive mutations. In order to identify cellular processes responsible for adaptive mutagenesis in eukaryotes, we study frameshift mutations occurring during auxotrophy-caused cell cycle arrest in the model organism Saccharomyces cerevisiae. Previous work has shown that an exposure of cells to UV irradiation during prolong...
Source: DNA Repair - November 10, 2009 Category: Genetics & Stem Cells Authors: Heidenreich E, Eisler H, Lengheimer T, Dorninger P, Steinboeck F Tags: DNA Repair (Amst) Source Type: journals

Interacting partners of the Tfb2 subunit from yeast TFIIH.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
TFIIH is an evolutionary conserved eukaryotic multi-protein complex composed of ten subunits. It is involved in transcription, cell cycle regulation, RNA splicing and the nucleotide excision DNA repair pathway (NER). Depending on the process in which it is functioning, the composition of TFIIH varies and activities of its subunits are differentially regulated. Here we focused on interplay between the Ssl2, Tfb2 and Tfb5 subunits of TFIIH from Saccharomyces cerevisiae. We found that Tfb2 bridges the Ssl2 helicase and the NER-specific Tfb5 subunit. Moreover, the Tfb5-interacting domain of Tfb2 also binds nucleic acids (N...
Source: DNA Repair - November 6, 2009 Category: Genetics & Stem Cells Authors: Kainov DE, Selth LA, Svejstrup JQ, Egly JM, Poterzsman A Tags: DNA Repair (Amst) Source Type: journals

Near-full-length REV3L appears to be a scarce maternal factor in Xenopus laevis eggs that changes qualitatively in early embryonic development.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report that Xenopus laevisREV3L encodes a 352-kDa protein that has high overall amino acid sequence similarity to its mammalian counterparts, and, for the first time in a vertebrate species, we have detected putative REV3L polypeptides of 300 and 340kDa in X. laevis oocytes. Only the 300-kDa form is stored in eggs, where its concentration of about 65pM is much lower than those of other replication and repair proteins including the accessory pol zeta subunit REV7. In fertilized eggs, the levels of this polypeptide did not change until neurula; the larger 340-kDa form first appeared at stages after gastrula, suggesting a ...
Source: DNA Repair - November 5, 2009 Category: Genetics & Stem Cells Authors: Ogawara D, Muroya T, Yamauchi K, Iwamoto TA, Yagi Y, Yamashita Y, Waga S, Akiyama M, Maki H Tags: DNA Repair (Amst) Source Type: journals

Divergent cellular phenotypes of human and mouse cells lacking the Werner syndrome RecQ helicase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Werner syndrome (WS) is a human autosomal recessive genetic instability and cancer predisposition syndrome with features of premature aging. Several genetically determined mouse models of WS have been generated, however, none develops features of premature aging or an elevated risk of neoplasia unless additional genetic perturbations are introduced. In order to determine whether differences in cellular phenotype could explain the discrepant phenotypes of Wrn(-/-) mice and WRN-deficient humans, we compared the cellular phenotype of newly derived Wrn(-/-) mouse primary fibroblasts with previous analyses of primary and tr...
Source: DNA Repair - November 4, 2009 Category: Genetics & Stem Cells Authors: Dhillon KK, Sidorova JM, Albertson TM, Anderson JB, Ladiges WC, Rabinovitch PS, Preston BD, Monnat RJ Tags: DNA Repair (Amst) Source Type: journals

The rad52-Y66A allele alters the choice of donor template during spontaneous chromosomal recombination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study we have used a rad52 hyper-recombination mutant, rad52-Y66A, to investigate the process of spontaneous heteroallelic recombination in the yeast Saccharomyces cerevisiae. We find that spontaneous recombination has different genetic requirements, depending on whether the recombination event occurs between chromosomes or between chromosome and plasmid sequences. The hyper-recombination phenotype of the rad52-Y66A mutation is epistatic with deletion of MRE11, which is required for establishment of DNA damage-induced cohesion. Moreover, single-cell analysis of strains expressing YFP-tagged Rad52-Y66A reveals a clo...
Source: DNA Repair - November 3, 2009 Category: Genetics & Stem Cells Authors: de Mayolo AA, Sunjevaric I, Reid R, Mortensen UH, Rothstein R, Lisby M Tags: DNA Repair (Amst) Source Type: journals

Dissection of Rad9 BRCT domain function in the mitotic checkpoint response to telomere uncapping.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In Saccharomyces cerevisiae, destabilizing telomeres, via inactivation of telomeric repeat binding factor Cdc13, induces a cell cycle checkpoint that arrests cells at the metaphase to anaphase transition-much like the response to an unrepaired DNA double strand break (DSB). Throughout the cell cycle, the multi-domain adaptor protein Rad9 is required for the activation of checkpoint effector kinase Rad53 in response to DSBs and is similarly necessary for checkpoint signaling in response to telomere uncapping. Rad53 activation in G1 and S phase depends on Rad9 association with modified chromatin adjacent to DSBs, which i...
Source: DNA Repair - October 30, 2009 Category: Genetics & Stem Cells Authors: Nnakwe CC, Altaf M, Côté J, Kron SJ Tags: DNA Repair (Amst) Source Type: journals

Specific targeted gene repair using single-stranded DNA oligonucleotides at an endogenous locus in mammalian cells uses homologous recombination.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The feasibility of introducing point mutations in vivo using single-stranded DNA oligonucleotides (ssON) has been demonstrated but the efficiency and mechanism remain elusive and potential side effects have not been fully evaluated. Understanding the mechanism behind this potential therapy may help its development. Here, we demonstrate the specific repair of an endogenous non-functional hprt gene by a ssON in mammalian cells, and show that the frequency of such an event is enhanced when cells are in S-phase of the cell cycle. A potential barrier in using ssONs as gene therapy could be non-targeted mutations or gene rea...
Source: DNA Repair - October 23, 2009 Category: Genetics & Stem Cells Authors: McLachlan J, Fernandez S, Helleday T, Bryant HE Tags: DNA Repair (Amst) Source Type: journals

A soluble RecN homologue provides means for biochemical and genetic analysis of DNA double-strand break repair in Escherichia coli.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
RecN is a highly conserved, SMC-like protein in bacteria. It plays an important role in the repair of DNA double-strand breaks and is therefore a key factor in maintaining genome integrity. The insolubility of Escherichia coli RecN has limited efforts to unravel its function. We overcame this limitation by replacing the resident coding sequence with that of Haemophilus influenzae RecN. The heterologous construct expresses Haemophilus RecN from the SOS-inducible E. coli promoter. The hybrid gene is fully functional, promoting survival after I-SceI induced DNA breakage, gamma irradiation or exposure to mitomycin C as eff...
Source: DNA Repair - October 18, 2009 Category: Genetics & Stem Cells Authors: Grove JI, Wood SR, Briggs GS, Oldham NJ, Lloyd RG Tags: DNA Repair (Amst) Source Type: journals

Electron microscopy of Xrcc4 and the DNA ligase IV-Xrcc4 DNA repair complex.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The DNA ligase IV-Xrcc4 complex is responsible for the ligation of broken DNA ends in the non-homologous end-joining (NHEJ) pathway of DNA double strand break repair in mammals. Mutations in DNA ligase IV (Lig4) lead to immunodeficiency and radiosensitivity in humans. Only partial structural information for Lig4 and Xrcc4 is available, while the structure of the full-length proteins and their arrangement within the Lig4-Xrcc4 complex is unknown. The C-terminal domain of Xrcc4, whose structure has not been solved, contains phosphorylation sites for DNA-PKcs and is phylogenetically conserved, indicative of a regulatory r...
Source: DNA Repair - October 14, 2009 Category: Genetics & Stem Cells Authors: Recuero-Checa MA, Doré AS, Arias-Palomo E, Rivera-Calzada A, Scheres SH, Maman JD, Pearl LH, Llorca O Tags: DNA Repair (Amst) Source Type: journals

Adenine removal activity and bacterial complementation with the human MutY homologue (MUTYH) and Y165C, G382D, P391L and Q324R variants associated with colorectal cancer.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, a kinetic analysis of the adenine glycosylase activity of MUTYH and several variants was undertaken using a correction for active fraction to control for differences due to overexpression and purification. Using these methods, the rate constants for steps involved in the adenine removal process were determined for the MAP variants Y165C, G382D, P391L and Q324R MUTYH. Under single-turnover conditions, the rate of adenine removal for these four variants was found to be 30-40% of WT MUTYH. In addition, the ability of MUTYH and the variants to suppress mutations and complement for the absence of MutY in Escheric...
Source: DNA Repair - October 13, 2009 Category: Genetics & Stem Cells Authors: Kundu S, Brinkmeyer MK, Livingston AL, David SS Tags: DNA Repair (Amst) Source Type: journals

Embryonic stem cells lacking the epigenetic regulator Cfp1 are hypersensitive to DNA-damaging agents and exhibit decreased Ape1/Ref-1 protein expression and endonuclease activity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Modulation of chromatin structure plays an important role in the recruitment and function of DNA repair proteins. CXXC finger protein 1 (Cfp1), encoded by the CXXC1 gene, is essential for mammalian development and is an important regulator of chromatin structure. Murine embryonic stem (ES) cells lacking Cfp1 (CXXC1(-/-)) are viable but demonstrate a dramatic decrease in cytosine methylation, altered histone methylation, and an inability to differentiate. We find that ES cells lacking Cfp1 are hypersensitive to a variety of DNA-damaging agents. In addition, CXXC1(-/-) ES cells accumulate more DNA damage and exhibit decr...
Source: DNA Repair - October 13, 2009 Category: Genetics & Stem Cells Authors: Tate CM, Fishel ML, Holleran JL, Egorin MJ, Skalnik DG Tags: DNA Repair (Amst) Source Type: journals

Mms1-Mms22 complex protects genome integrity in Schizosaccharomyces pombe.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mms1 and Mms22 are subunits of an Rtt101-based E3 ubiquitin ligase required for replication of damaged DNA templates in Saccharomyces cerevisiae. The function and evolutionary conservation of this DNA repair module are unknown. Here we report the characterization of an Mms1 ortholog in Schizosaccharomyces pombe. Fission yeast Mms1 was discovered through its physical association with S. pombe Mms22 (also known as Mus7). Loss of S. pombe Mms1 results in the accumulation of spontaneous DNA damage, mitotic delay, and hypersensitivity to genotoxins such as camptothecin that perturb replisome progression. Homologous recombin...
Source: DNA Repair - October 8, 2009 Category: Genetics & Stem Cells Authors: Dovey CL, Aslanian A, Sofueva S, Yates JR, Russell P Tags: DNA Repair (Amst) Source Type: journals

Apex1 can cleave complex clustered DNA lesions in cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Current data indicate that clustered DNA damage generated by ionizing radiation contains 2-5 damages within 20bps. The complexity of clustered damage is also believed to increase as the linear energy transfer of the radiation increases. Complex lesions are therefore biologically relevant especially with the use of carbon ion beam therapy to treat cancer. Since two closely opposed AP site analogs (furans) are converted to a double strand break (DSB) in cells, we hypothesized that breakage could be compromised by increasing the complexity of the cluster. We have examined the repair of clusters containing three and four l...
Source: DNA Repair - September 29, 2009 Category: Genetics & Stem Cells Authors: Malyarchuk S, Castore R, Harrison L Tags: DNA Repair (Amst) Source Type: journals

A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In fungal species, lysine 56 of newly synthesized histone H3 molecules is modified by the acetyltransferase Rtt109, which promotes resistance to genotoxic agents. To further explore how H3 K56ac contributes to genome stability, we conducted screens for suppressors of the DNA damage sensitivity of budding yeast rtt109Delta mutants. We recovered a single extragenic suppressor mutation that efficiently restored damage resistance. The suppressor is a point mutation in the histone H3 gene HHT2, and converts lysine 56 to glutamic acid. In some ways, K56E mimics K56ac, because it suppresses other mutations that interfere with...
Source: DNA Repair - September 28, 2009 Category: Genetics & Stem Cells Authors: Erkmann JA, Kaufman PD Tags: DNA Repair (Amst) Source Type: journals

Mammalian polymerase zeta is essential for post-replication repair of UV-induced DNA lesions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
DNA polymerase zeta is believed to be an essential constituent of DNA damage tolerance, comprising several pathways that allow the replication of DNA templates containing unrepaired damage. We wanted to better define the role of polymerase zeta in DNA damage tolerance in mammalian cells. To this aim we have investigated replication of ultraviolet light-damaged DNA templates in mouse embryonic fibroblasts deficient for Rev3, the catalytic subunit of polymerase zeta. We found that Rev3 is important for a post-replication repair pathway of helix-distorting [6-4]pyrimidine-pyrimidone photoproducts and, to a lesser extent, ...
Source: DNA Repair - September 25, 2009 Category: Genetics & Stem Cells Authors: Jansen JG, Tsaalbi-Shtylik A, Hendriks G, Verspuy J, Gali H, Haracska L, de Wind N Tags: DNA Repair (Amst) Source Type: journals

Polk mutant mice have a spontaneous mutator phenotype.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mice defective for the Polk gene, which encodes DNA polymerase kappa, are viable and do not manifest obvious phenotypes. The present studies document a spontaneous mutator phenotype in Polk(-/-) mice. The initial indication of enhanced spontaneous mutations in these mice came from the serendipitous observation of a postulated founder mutation that manifested in multiple disease states among a cohort of mice comprising all three possible Polk genotypes. Polk(-/-) and isogenic wild-type controls carrying a reporter transgene (the lambda-phage cII gene) were used for subsequent quantitative and qualitative studies on muta...
Source: DNA Repair - September 24, 2009 Category: Genetics & Stem Cells Authors: Stancel JN, McDaniel LD, Velasco S, Richardson J, Guo C, Friedberg EC Tags: DNA Repair (Amst) Source Type: journals

Mouse models for ATR deficiency.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
ATM and ATR orchestrate overlapping DNA damage responses in reply to different forms of DNA strand discontinuities. But, knockout mouse models suggest that ATR is essential for viability in contrast to ATM. Recently, more sophisticated mouse models have been published including a conditional ATR-knockdown system and by modelling the human ATR-Seckel syndrome-causative mutation. Here, I will overview and contrast these models highlighting the advances both represent in our understanding of how defects in the ATR-dependent DNA damage response can impact on normal development, tissue homeostasis, ageing and cancer. PM...
Source: DNA Repair - September 23, 2009 Category: Genetics & Stem Cells Authors: O'Driscoll M Tags: DNA Repair (Amst) Source Type: journals

NBS1 cooperates with homologous recombination to counteract chromosome breakage during replication.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Nijmegen breakage syndrome (NBS) is characterized by genome instability and cancer predisposition. NBS patients contain a mutation in the NBS1 gene, which encodes the NBS1 component of the DNA double-strand break (DSB) response complex MRE11/RAD50/NBS1. To investigate the NBS phenotype in more detail, we combined the mouse mimic of the most common patient mutation (Nbs1(DeltaB/DeltaB)) with a Rad54 null mutation, which diminishes homologous recombination. Double mutant cells were particularly sensitive to treatments that cause single strand breaks (SSBs), presumably because these SSBs can be converted into detrimental ...
Source: DNA Repair - September 23, 2009 Category: Genetics & Stem Cells Authors: Brugmans L, Verkaik NS, Kunen M, van Drunen E, Williams BR, Petrini JH, Kanaar R, Essers J, van Gent DC Tags: DNA Repair (Amst) Source Type: journals

Deficiency of the oxidative damage-specific DNA glycosylase NEIL1 leads to reduced germinal center B cell expansion.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mammalian cells possess multiple DNA glycosylases, including OGG1, NTH1, NEIL1, NEIL2 and NEIL3, for the repair of oxidative DNA damage. Among these, NEIL1 and NEIL2 are able to excise oxidized bases on single stranded or bubble-structured DNA and has been implicated in repair of oxidative damage associated with DNA replication or transcription. We found that Neil1 was highly constitutively expressed in the germinal center (GC) B cells, a rapidly dividing cell population that is undergoing immunoglobulin (Ig) gene hypermutation and isotype switching. While Neil1(-/-) mice exhibited normal B and T cell development and m...
Source: DNA Repair - September 22, 2009 Category: Genetics & Stem Cells Authors: Mori H, Ouchida R, Hijikata A, Kitamura H, Ohara O, Li Y, Gao X, Yasui A, Lloyd RS, Wang JY Tags: DNA Repair (Amst) Source Type: journals

The unstructured C-terminal extension of UvrD interacts with UvrB, but is dispensable for nucleotide excision repair.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We examined this interaction using 2-hybrid analysis and surface plasmon resonance spectroscopy, and found that the N-terminal domain and the unstructured region at the C-terminus of UvrD interact with UvrB. We analysed the properties of a truncated UvrD protein that lacked the unstructured C-terminal region and found that it showed a diminished affinity for single-stranded DNA, but retained the ability to displace both UvrC and the lesion-containing oligonucleotide from a post-incision nucleotide excision repair complex. The interaction of the C-terminal region of UvrD with UvrB is therefore not an essential feature of th...
Source: DNA Repair - September 14, 2009 Category: Genetics & Stem Cells Authors: Manelyte L, Guy CP, Smith RM, Dillingham MS, McGlynn P, Savery NJ Tags: DNA Repair (Amst) Source Type: journals

DNA polymerase beta and PARP activities in base excision repair in living cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
To examine base excision repair (BER) capacity in the context of living cells, we developed and applied a plasmid-based reporter assay. Non-replicating plasmids containing unique DNA base lesions were designed to express luciferase only after lesion repair had occurred, and luciferase expression in transfected cells was measured continuously during a repair period of 14h. Two types of DNA lesions were examined: uracil opposite T reflecting repair primarily by the single-nucleotide BER sub-pathway, and the abasic site analogue tetrahydrofuran (THF) opposite C reflecting repair by long-patch BER. We found that the repair...
Source: DNA Repair - September 10, 2009 Category: Genetics & Stem Cells Authors: Masaoka A, Horton JK, Beard WA, Wilson SH Tags: DNA Repair (Amst) Source Type: journals

Structural basis for the lack of opposite base specificity of Clostridium acetobutylicum 8-oxoguanine DNA glycosylase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
7,8-Dihydro-8-oxoguanine (8-oxoG) is the major oxidative product of guanine and the most prevalent base lesion observed in DNA molecules. Because 8-oxoG has the capability to form a Hoogsteen pair with adenine (8-oxoG:A) in addition to a normal Watson-Crick pair with cytosine (8-oxoG:C), this lesion can lead to a G:C-->T:A transversion after replication. However, 8-oxoG is recognized and excised by the 8-oxoguanine DNA glycosylase (Ogg) of the base excision repair pathway. Members of the Ogg1 family usually display a strong preference for a C opposite the lesion. In contrast, the atypical Ogg1 from Clostridium actet...
Source: DNA Repair - September 8, 2009 Category: Genetics & Stem Cells Authors: Faucher F, Wallace SS, Doublié S Tags: DNA Repair (Amst) Source Type: journals

The roles of SbcCD and RNaseE in the transcription of GAA.TTC repeats in Escherichia coli.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Expansion of GAA.TTC repeats in FXN gene is associated with decreased frataxin production in Frederich's ataxia patients. To study this effect, we have engineered a set of GAA.TTC repeats in the EcoRI site of lacZ gene of plasmid pUC18 as part of the transcription template of the lacZ gene, while keeping its ORF unchanged. The effects of the GAA.TTC repeats on the lacZ expression were investigated in Escherichia coli JM83 and its mutants deficiency in RNA processing, homologous recombination and DNA repair. We found that transcriptions of the GAA strand with different sizes and organizations displayed normal alpha-comp...
Source: DNA Repair - September 2, 2009 Category: Genetics & Stem Cells Authors: Pan X, Ding Y, Shi L Tags: DNA Repair (Amst) Source Type: journals

Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress: Use of APE1 small molecule inhibitors to delineate APE1 functions.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H(2)O(2). However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 tha...
Source: DNA Repair - August 30, 2009 Category: Genetics & Stem Cells Authors: Jiang Y, Guo C, Fishel ML, Wang ZY, Vasko MR, Kelley MR Tags: DNA Repair (Amst) Source Type: journals

PARP inhibition during alkylation-induced genotoxic stress signals a cell cycle checkpoint response mediated by ATM.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
By limiting cell cycle progression following detection of DNA damage, checkpoints are critical for cell survival and genome stability. Methylated DNA damage, when combined with inhibition of PARP activity, results in an ATR-dependent S phase delay of the cell cycle. Here, we demonstrate that another checkpoint kinase, ATM, also is involved in the DNA damage response following treatment with a sub-lethal concentration of MMS combined with the PARP inhibitor 4-AN. Both ATM and PARP activities are important for moderating cellular sensitivity to MMS. Loss of ATM activity, or that of its downstream effector Chk2, limited t...
Source: DNA Repair - August 27, 2009 Category: Genetics & Stem Cells Authors: Carrozza MJ, Stefanick DF, Horton JK, Kedar PS, Wilson SH Tags: DNA Repair (Amst) Source Type: journals

Activity of ribonucleotide reductase helps determine how cells repair DNA double strand breaks.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Mammalian cells can choose either nonhomologous end joining (NHEJ) or homologous recombination (HR) for repair of chromosome breaks. Of these two pathways, HR alone requires extensive DNA synthesis and thus abundant synthesis precursors (dNTPs). We address here if this differing requirement for dNTPs helps determine how cells choose a repair pathway. Cellular dNTP pools are regulated primarily by changes in ribonucleotide reductase activity. We show that an inhibitor of ribonucleotide reductase (hydroxyurea) hypersensitizes NHEJ-deficient cells, but not wild type or HR-deficient cells, to chromosome breaks introduced b...
Source: DNA Repair - August 24, 2009 Category: Genetics & Stem Cells Authors: Burkhalter MD, Roberts SA, Havener JM, Ramsden DA Tags: DNA Repair (Amst) Source Type: journals

PIKK-dependent phosphorylation of Mre11 induces MRN complex inactivation by disassembly from chromatin.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The role of Mre11 phosphorylation in the cellular response to DNA double-strand breaks (DSBs) is not well understood. Here, we show that phosphorylation of Mre11 at SQ/TQ motifs by PIKKs (PI3 Kinase-related Kinases) induces MRN (Mre11-Rad50-Nbs1) complex dissociation from chromatin by reducing Mre11 affinity for DNA. Whereas phosphorylation of Mre11 at these residues is not required for DSB-induced ATM (Ataxia-Telangiectasia mutated) activation, abrogation of Mre11 dephosphorylation impairs ATM signaling. Our study provides a functional characterization of the DNA damage-induced Mre11 phosphorylation, and suggests that...
Source: DNA Repair - August 23, 2009 Category: Genetics & Stem Cells Authors: Di Virgilio M, Ying CY, Gautier J Tags: DNA Repair (Amst) Source Type: journals

Evidence for a role of FEN1 in maintaining mitochondrial DNA integrity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we analyzed the effects of deleting the yeast FEN1 homolog, RAD27, on mtDNA stability in Saccharomyces cerevisiae. Our findings demonstrate that Rad27p/FEN1 is localized in the mitochondrial compartment of both yeast and mice and that Rad27p has a significant role in maintaining mtDNA integrity. PMID: 19699691 [PubMed - as supplied by publisher] (Source: DNA Repair)
Source: DNA Repair - August 19, 2009 Category: Genetics & Stem Cells Authors: Kalifa L, Beutner G, Phadnis N, Sheu SS, Sia EA Tags: DNA Repair (Amst) Source Type: journals

Regulation of repair choice: Cdk1 suppresses recruitment of end joining factors at DNA breaks.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Cell cycle plays a crucial role in regulating the pathway used to repair DNA double-strand breaks (DSBs). In Saccharomyces cerevisiae, homologous recombination is primarily limited to non-G(1) cells as the formation of recombinogenic single-stranded DNA requires CDK1-dependent 5' to 3' resection of DNA ends. However, the effect of cell cycle on non-homologous end joining (NHEJ) is not yet clearly defined. Using an assay to quantitatively measure the contributions of each repair pathway to repair product formation and cellular survival after DSB induction, we found that NHEJ is most efficient at G(1), and markedly repre...
Source: DNA Repair - August 19, 2009 Category: Genetics & Stem Cells Authors: Zhang Y, Shim EY, Davis M, Lee SE Tags: DNA Repair (Amst) Source Type: journals

NUDT5 hydrolyzes oxidized deoxyribonucleoside diphosphates with broad substrate specificity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The human NUDT5 protein catalyzes the hydrolysis of 8-hydroxy-dGDP. To examine its substrate specificity, four oxidized deoxyribonucleotides (2-hydroxy-dADP, 8-hydroxy-dADP, 5-formyl-dUDP, and 5-hydroxy-dCDP) were incubated with the NUDT5 protein. Interestingly, all of the nucleotides, except for 5-hydroxy-dCDP, were hydrolyzed with various efficiencies. The kinetic parameters indicated that 8-hydroxy-dADP was hydrolyzed as efficiently as 8-hydroxy-dGDP. The hydrolyzing activities for their triphosphate counterparts were quite weak. These results suggest that the NUDT5 protein eliminates various oxidized deoxyribonucle...
Source: DNA Repair - August 19, 2009 Category: Genetics & Stem Cells Authors: Kamiya H, Hori M, Arimori T, Sekiguchi M, Yamagata Y, Harashima H Tags: DNA Repair (Amst) Source Type: journals

DNA lesions sequestered in micronuclei induce a local defective-damage response.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Micronuclei are good markers of chromosome instability and, among other disturbances, are closely related to double-strand break induction. The ability of DNA lesions sequestered in the micronuclear bodies to activate the complex damage-signalling network is highly controversial since some repair factors have not been consistently detected inside micronuclei. In order to better understand the efficiency of the response induced by micronuclear DNA damage, we have analyzed the presence of DNA damage-response factors and DNA degradation markers in these structures. Radiation-induced DNA double-strand breaks produce a modi...
Source: DNA Repair - August 12, 2009 Category: Genetics & Stem Cells Authors: Terradas M, Martín M, Tusell L, Genescà A Tags: DNA Repair (Amst) Source Type: journals

[In Process Citation]Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Authors: PMID: 19631183 [PubMed - in process] (Source: DNA Repair)
Source: DNA Repair - July 30, 2009 Category: Genetics & Stem Cells Tags: DNA Repair (Amst) Source Type: journals

Cellular pathways for DNA repair and damage tolerance of formaldehyde-induced DNA-protein crosslinks.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Although it is well established that DNA-protein crosslinks are formed as a consequence of cellular exposure to agents such as formaldehyde, transplatin, ionizing and ultraviolet radiation, the biochemical pathways that promote cellular survival via repair or tolerance of these lesions are poorly understood. To investigate the mechanisms that function to limit DNA-protein crosslink-induced cytotoxicity, the Saccharomyces cerevisiae non-essential gene deletion library was screened for increased sensitivity to formaldehyde exposure. Following low dose, chronic exposure, strains containing deletions in genes mediating hom...
Source: DNA Repair - July 19, 2009 Category: Genetics & Stem Cells Authors: de Graaf B, Clore A, McCullough AK Tags: DNA Repair (Amst) Source Type: journals

TREX1 acts in degrading damaged DNA from drug-treated tumor cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report that the expression and localization of TREX1 are cell-type dependent. Camptothecin and other DNA damaging agents induced both TREX1 protein and its mRNA in a dose- and time-dependent manner. Using a TREX1-inducible cell line, we performed clonogenic assays and found no change in sensitivity of the cells to the agents upon TREX1 induction, suggesting that TREX1 may not play a role in DNA repair or drug sensitivity. Nevertheless, TREX1 serves as a key enzyme in the degradation of DNA from dying cells leading to less cellular DNA. Ubiquitously expressed in normal tissues, TREX1 may act in degrading DNA in all cell ...
Source: DNA Repair - July 16, 2009 Category: Genetics & Stem Cells Authors: Wang CJ, Lam W, Bussom S, Chang HM, Cheng YC Tags: DNA Repair (Amst) Source Type: journals

Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N(6)-ethenoadenine (varepsilonA) from varepsilonA-containing oligonucleotide (varepsilonA-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (K(D)) towards MPG,...
Source: DNA Repair - July 15, 2009 Category: Genetics & Stem Cells Authors: Adhikari S, Uren A, Roy R Tags: DNA Repair (Amst) Source Type: journals

Repair activities of human 8-oxoguanine DNA glycosylase are stimulated by the interaction with human checkpoint sensor Rad9-Rad1-Hus1 complex.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we identified a novel interaction between hOGG1 and human 9-1-1, and investigated the functional consequences of this interaction. Co-immunoprecipitation assays using transiently transfected HEK293 cells demonstrated an interaction between hOGG1 and the 9-1-1 proteins. Subsequently, GST pull-down assays using bacterially expressed and purified hOGG1-His and GST-fused 9-1-1 subunits (GST-hRad9, GST-hRad1, and GST-hHus1) demonstrated that hOGG1 interacted directly with the individual subunits of the human 9-1-1 complex. In vitro excision assay, which employed a DNA duplex containing an 8-oxoG/C mismatch, showe...
Source: DNA Repair - July 14, 2009 Category: Genetics & Stem Cells Authors: Park MJ, Park JH, Hahm SH, Ko SI, Lee YR, Chung JH, Cho Y, Kang LW, Han YS Tags: DNA Repair (Amst) Source Type: journals

Characterization of DNA damage-dependent cell cycle checkpoints in a menin-deficient model.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we found that loss of Men1 in mouse embryonic fibroblasts caused abrogation of the G1/S and intra-S checkpoints following ionizing radiation. The cyclin-dependent kinase inhibitor, p21, failed to be upregulated in the mutant although upstream checkpoint signaling remained intact. Menin localized to the p21 promoter in a DNA damage-dependent manner. The MLL histone methyltransferase, a positive transcriptional regulator, bound to the same region in the presence of menin but not in Men1(-/-) cells. Finally, p53 retained damage-responsive binding to the p21 promoter in the Men1 mutant. These data indicate that ...
Source: DNA Repair - July 13, 2009 Category: Genetics & Stem Cells Authors: Kottemann MC, Bale AE Tags: DNA Repair (Amst) Source Type: journals

Localization of X-ray cross complementing gene 1 protein in the nuclear matrix is controlled by casein kinase II-dependent phosphorylation in response to oxidative damage.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Base excision repair/single strand break repair (BER/SSBR) of damaged DNA is a highly efficient process. X-ray cross complementing protein 1 (XRCC1) functions as a key scaffold protein for BER/SSBR factors. Recent work has shown that XRCC1 forms dense foci at sites of DNA damage in a manner dependent on casein kinase II (CK2) phosphorylation. To investigate the mechanism underlying foci formation, we analyzed the subnuclear localization and phosphorylation status of XRCC1 during the repair process by biochemical fractionation of HeLa cellular proteins. The localization was also verified by in situ extraction of the fix...
Source: DNA Repair - July 8, 2009 Category: Genetics & Stem Cells Authors: Kubota Y, Takanami T, Higashitani A, Horiuchi S Tags: DNA Repair (Amst) Source Type: journals

Distinct kinetics of human DNA ligases I, IIIalpha, IIIbeta, and IV reveal direct DNA sensing ability and differential physiological functions in DNA repair.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we developed and optimized a fluorescence-based DNA ligation assay to characterize the activities of purified hLigs. Although hLigI joins DNA nicks, it has no detectable activity on linear duplex DNA substrates with short, cohesive single-strand ends. By contrast, hLigIIIbeta and the hLigIIIalpha/XRCC1 and hLigIV/XRCC4 complexes are active on both nicked and linear duplex DNA substrates. Surprisingly, hLigIV/XRCC4, which is a key component of the major non-homologous end joining (NHEJ) pathway, is significantly less active than hLigIII on a linear duplex DNA substrate. Notably, hLigIV/XRCC4 molecules only ca...
Source: DNA Repair - July 6, 2009 Category: Genetics & Stem Cells Authors: Chen X, Ballin JD, Della-Maria J, Tsai MS, White EJ, Tomkinson AE, Wilson GM Tags: DNA Repair (Amst) Source Type: journals

The role of the retinoblastoma/E2F1 tumor suppressor pathway in the lesion recognition step of nucleotide excision repair.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The retinoblastoma Rb/E2F tumor suppressor pathway plays a major role in the regulation of mammalian cell cycle progression. The pRb protein, along with closely related proteins p107 and p130, exerts its anti-proliferative effects by binding to the E2F family of transcription factors known to regulate essential genes throughout the cell cycle. We sought to investigate the role of the Rb/E2F1 pathway in the lesion recognition step of nucleotide excision repair (NER) in mouse embryonic fibroblasts (MEFs). Rb-/-, p107-/-, p130-/- MEFs repaired both cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs) at hig...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Lin PS, McPherson LA, Chen AY, Sage J, Ford JM Tags: DNA Repair (Amst) Source Type: journals

Construction of a circular single-stranded DNA template containing a defined lesion.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
We report a concise and efficient method to make a circular single-stranded DNA containing a defined DNA lesion. In this protocol, phagemid DNA containing Uracil is used as a template to synthesize a complementary DNA strand using T7 DNA polymerase and an oligonucleotide primer including a site-specific DNA lesion. The ligated lesion-containing strand can be recovered after the phage-derived template DNA is degraded by treatment with E. coli Uracil DNA glycosylase and Exonucleases I and III. The resulting product is a circular single-stranded DNA containing a defined DNA lesion suitable for in vitro translesion replication...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Karata K, Vidal AE, Woodgate R Tags: DNA Repair (Amst) Source Type: journals

DNA damage responses in Drosophila nbs mutants with reduced or altered NBS function.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The MRN complex, composed of MRE11, RAD50 and NBS, plays important roles in responding to DNA double-strand breaks (DSBs). In metazoans, functional studies of genes encoding these proteins have been challenging because complete loss-of-function mutations are lethal at the organismal level and because NBS has multiple functions in DNA damage responses. To study functions of Drosophila NBS in DNA damage responses, we used a separation-of-function mutation that causes loss of the forkhead-associated (FHA) domain. Loss of the FHA domain resulted in hypersensitivity to ionizing radiation and defects in gap repair by homolog...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Mukherjee S, Lafave MC, Sekelsky J Tags: DNA Repair (Amst) Source Type: journals

Accessibility of chromosomal recombination breaks in nuclei of wild-type and DNA-PKcs-deficient cells.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
V(D)J recombination is a highly regulated process, proceeding from a site-specific cleavage to an imprecise end joining. After the DNA excision catalyzed by the recombinase encoded by recombination activating genes 1 and 2 (RAG1/2), newly generated recombination ends are believed held by a post-cleavage complex (PC) consisting of RAG1/2 proteins, and are subsequently resolved by non-homologous end joining (NHEJ) machinery. The relay of these ends from PC to NHEJ remains elusive. It has been speculated that NHEJ factors modify the RAG1/2-PC to gain access to the ends or act on free ends after the disassembly of the PC. ...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Franco D, Chang Y Tags: DNA Repair (Amst) Source Type: journals

Mutational studies of Pa-AGOG DNA glycosylase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
In this study, we made quantitative measurements of the DNA glycosylase activity of Pa-AGOG wild type and some engineered variants under single turnover conditions. The mutagenesis study includes residues Trp222 (W222A and W222F), Trp69 (W69F), Gln31 (Q31S) and Lys147 (K147Q) all of which are involved in GO recognition and Asp172 (D172N and D172Q) and Lys140 (K140Q) that are involved in catalysis. Pa-AGOG prefers GO/G mispairs for both base excision and base excision/beta-lyase activities. The mutagenesis studies show that base-stacking between GO and Trp222 is very important for recognition. The contact between Trp69 and ...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Lingaraju GM, Prota AE, Winkler FK Tags: DNA Repair (Amst) Source Type: journals

Overexpression of transcription factor AP-2 stimulates the P(A) promoter of the human uracil-DNA glycosylase (UNG) gene through a mechanism involving derepression.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
The P(A) promoter in the human uracil-DNA glycosylase gene (UNG) directs expression of the nuclear form (UNG2) of UNG proteins. Using a combination of promoter deletion and mutation analyses, and transient transfection of HeLa cells, we show that repressor and derepressor activities are contained within the region of DNA marked by P(A). Footprinting analysis and electrophoretic mobility shift assays of P(A) and putative AP-2 binding regions with HeLa cell nuclear extract and recombinant AP-2alpha protein indicate that AP-2 transcription factors are central in the regulated expression of UNG2 mRNA. Chromatin immunopreci...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Aas PA, Peña-Diaz J, Liabakk NB, Krokan HE, Skorpen F Tags: DNA Repair (Amst) Source Type: journals

Extracts of proliferating and non-proliferating human cells display different base excision pathways and repair fidelity.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Base excision repair (BER) of damaged or inappropriate bases in DNA has been reported to take place by single nucleotide insertion or through incorporation of several nucleotides, termed short-patch and long-patch repair, respectively. We found that extracts from proliferating and non-proliferating cells both had capacity for single- and two-nucleotide insertion BER activity. However, patch size longer than two nucleotides was only detected in extracts from proliferating cells. Relative to extracts from proliferating cells, extracts from non-proliferating cells had approximately two-fold higher concentration of POLbeta...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Akbari M, Peña-Diaz J, Andersen S, Liabakk NB, Otterlei M, Krokan HE Tags: DNA Repair (Amst) Source Type: journals

Histone H2A.X Tyr142 phosphorylation: A novel sWItCH for apoptosis?Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Histone H2A.X phosphorylation on Ser139 in response to DNA damage is the major signal for the assembly of the so-called gammaH2A.X chromatin domain, a region surrounding an unrepaired DNA double-strand break that is characterized by the accumulation of a large number of DNA damage response proteins. However, it is not yet clear how this event is regulated in space and time. The recent discovery of H2A.X Tyr142 phosphorylation by the WICH complex and its dephosphorylation by the EYA1/3 phosphatases may provide substantial novel insight into this process. WSTF, a subunit of the WICH complex bears a novel kinase domain at...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Stucki M Tags: DNA Repair (Amst) Source Type: journals

Human HMGB1 directly facilitates interactions between nucleotide excision repair proteins on triplex-directed psoralen interstrand crosslinks.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Psoralen is a chemotherapeutic agent that acts by producing DNA interstrand crosslinks (ICLs), which are especially cytotoxic and mutagenic because their complex chemical nature makes them difficult to repair. Proteins from multiple repair pathways, including nucleotide excision repair (NER), are involved in their removal in mammalian cells, but the exact nature of their repair is poorly understood. We have shown previously that HMGB1, a protein involved in chromatin structure, transcriptional regulation, and inflammation, can bind cooperatively to triplex-directed psoralen ICLs with RPA, and that mammalian cells lacki...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Lange SS, Reddy MC, Vasquez KM Tags: DNA Repair (Amst) Source Type: journals

Purification and characterization of Caenorhabditis elegans NTH, a homolog of human endonuclease III: Essential role of N-terminal region.Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Oxidatively damaged bases in DNA cause many types of deleterious effects. The main enzyme that removes such lesions is DNA glycosylase, and accordingly, DNA glycosylase plays an important role in genome stability. Recently, a relationship between DNA glycosylases and aging has been suggested, but it remains controversial. Here, we investigated DNA glycosylases of C. elegans, which is a useful model organism for studying aging. We firstly identified a C. elegans homolog of endonuclease III (NTH), which is a well-conserved DNA glycosylase for oxidatively damaged pyrimidine bases, based on the activity and homology. Blast...
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Authors: Morinaga H, Yonekura S, Nakamura N, Sugiyama H, Yonei S, Zhang-Akiyama QM Tags: DNA Repair (Amst) Source Type: journals

[In Process Citation]Email this article to a colleague. Save this article to My Clippings. Discuss or comment on this article.
Authors: PMID: 19539240 [PubMed - in process] (Source: DNA Repair)
Source: DNA Repair - June 28, 2009 Category: Genetics & Stem Cells Tags: DNA Repair (Amst) Source Type: journals