| 1. |
- Blundred, Rachel, et al.
(författare)
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Human RECQL5 overcomes thymidine-induced replication stress.
- 2010
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 9:9, s. 964-75
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Tidskriftsartikel (refereegranskat)abstract
- Accurate DNA replication is essential to genome integrity and is controlled by five human RecQ helicases, of which at least three prevent cancer and ageing. Here, we have studied the role of RECQL5, which is the least characterised of the five human RecQ helicases. We demonstrate that overexpressed RECQL5 promotes survival during thymidine-induced slowing of replication forks in human cells. The RECQL5 protein relocates specifically to stalled replication forks and suppresses thymidine-induced RPA foci, CHK1 signalling, homologous recombination and gammaH2AX activation. It is unlikely that RECQL5 promotes survival through translesion synthesis as PCNA ubiquitylation is also reduced. Interestingly, we also found that overexpressing RECQL5 relieves cells of the cell cycle arrest normally imposed by thymidine, but without causing mutations. In conclusion, we propose that RECQL5 stabilises the replication fork allowing replication to overcome the effects of thymidine and complete the cell cycle.
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| 2. |
- Bochman, Matthew L, et al.
(författare)
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Unwinding the functions of the Pif1 family helicases
- 2010
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 9:3, s. 237-249
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Tidskriftsartikel (refereegranskat)abstract
- Helicases are ubiquitous enzymes found in all organisms that are necessary for all (or virtually all) aspects of nucleic acid metabolism. The Pif1 helicase family is a group of 5'-->3' directed, ATP-dependent, super family IB helicases found in nearly all eukaryotes. Here, we review the discovery, evolution, and what is currently known about these enzymes in Saccharomyces cerevisiae (ScPif1 and ScRrm3), Schizosaccharomyces pombe (SpPfh1), Trypanosoma brucei (TbPIF1, 2, 5, and 8), mice (mPif1), and humans (hPif1). Pif1 helicases variously affect telomeric, ribosomal, and mitochondrial DNA replication, as well as Okazaki fragment maturation, and in at least some cases affect these processes by using their helicase activity to disrupt stable nucleoprotein complexes. While the functions of these enzymes vary within and between organisms, it is evident that Pif1 family helicases are crucial for both nuclear and mitochondrial genome maintenance.
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| 3. |
- Chen, Jiang, et al.
(författare)
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A catalytic and non-catalytic role for the Yen1 nuclease in maintaining genome integrity in Kluyveromyces lactis
- 2012
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 11:10, s. 833-843
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Tidskriftsartikel (refereegranskat)abstract
- Yen1 is a nuclease identified in Saccharomyces cerevisiae that cleaves the Holliday junction (HJ) intermediate formed during homologous recombination. Alternative routes to disjoin HJs are performed by the Mus81/Mms4- and Sgs1/Top3/Rmi1-complexes. Here, we investigate the role of the Yen1 protein in the yeast Kluyveromyces lactis. We demonstrate that both yen1 mus81 and yen1 sgs1 double mutants displayed negative genetic interactions in the presence of DNA-damaging chemicals. To test if these phenotypes required the catalytic activity of Yen1, we introduced point mutations targeting the catalytic site of Yen1, which abolished the nuclease activity in vitro. Remarkably, catalytically inactive Yen1 did not exacerbate the hydroxyurea sensitivity of the sgs1Δ strain, which the yen1Δ allele did. In addition, overexpression of catalytically inactive Yen1 partially rescued the DNA damage sensitivity of both mus81 and sgs1 mutant strains albeit less efficiently than WT Yen1. These results suggest that Yen1 serves both a catalytic and non-catalytic role in its redundant function with Mus81 and Sgs1. Diploids lacking Mus81 had a severe defect in sporulation efficiency and crossover frequency, but diploids lacking both Mus81 and Yen1 showed no further reduction in spore formation. Hence, Yen1 had no evident role in meiosis. However, overexpression of WT Yen1, but not catalytically inactive Yen1 partially rescued the crossover defect in mus81/mus81 mutant diploids. Yen1 is a member of the RAD2/XPG-family of nucleases, but genetic analyses revealed no genetic interaction between yen1 and other family members (rad2, exo1 and rad27). In addition, yen1 mutants had normal nonhomologous end-joining efficiency. We discuss the similarities and differences between K. lactis Yen1 and Yen1/GEN1 from other organisms.
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| 4. |
- Fahrer, Jörg, et al.
(författare)
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Cytolethal distending toxin (CDT) is a radiomimetic agent and induces persistent levels of DNA double-strand breaks in human fibroblasts
- 2014
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Ingår i: DNA Repair. - : Elsevier. - 1568-7864 .- 1568-7856. ; 18, s. 31-43
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Tidskriftsartikel (refereegranskat)abstract
- Cytolethal distending toxin (CDT) is a unique genotoxin produced by several pathogenic bacteria. The tripartite protein toxin is internalized into mammalian cells via endocytosis followed by retrograde transport to the ER. Upon translocation into the nucleus, CDT catalyzes the formation of DNA double-strand breaks (DSBs) due to its intrinsic endonuclease activity. In the present study, we compared the DNA damage response (DDR) in human fibroblasts triggered by recombinant CDT to that of ionizing radiation (IR), a well-known DSB inducer. Furthermore, we dissected the pathways involved in the detection and repair of CDT-induced DNA lesions. qRT-PCR array-based mRNA and western blot analyses showed a partial overlap in the DDR pattern elicited by CDT and IR, with strong activation of both the ATM-Chk2 and the ATR-Chk1 axis. In line with its in vitro DNase I-like activity on plasmid DNA, neutral and alkaline Comet assay revealed predominant induction of DSBs in CDT-treated fibroblasts, whereas irradiation of cells generated higher amounts of SSBs and alkali-labile sites. Using confocal microscopy, the dynamics of the DSB surrogate marker γ-H2AX was monitored after pulse treatment with CDT or IR. In contrast to the fast induction and disappearance of γ-H2AX-foci observed in irradiated cells, the number of γ-H2AX-foci induced by CDT were formed with a delay and persisted. 53BP1 foci were also generated following CDT treatment and co-localized with γ-H2AX foci. We further demonstrated that ATM-deficient cells are very sensitive to CDT-induced DNA damage as reflected by increased cell death rates with concomitant cleavage of caspase-3 and PARP-1. Finally, we provided novel evidence that both homologous recombination (HR) and non-homologous end joining (NHEJ) protect against CDT-elicited DSBs. In conclusion, the findings suggest that CDT functions as a radiomimetic agent and, therefore, is an attractive tool for selectively inducing persistent levels of DSBs and unveiling the associated cellular responses.
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| 5. |
- Lagerqvist, Anne, et al.
(författare)
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DNA repair and replication influence the number of mutations per adduct of polycyclic aromatic hydrocarbons in mammalian cells
- 2011
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 10:8, s. 877-886
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Tidskriftsartikel (refereegranskat)abstract
- Polycyclic aromatic hydrocarbons (PAH) are an important class of environmental contaminants many of which require metabolic activation to DNA-reactive bay or fjord region diolepoxides (DE) in order to exert their mutagenic and carcinogenic effects. In this study, the mutagenicity of the bay region diolepoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and ()-anti-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrodibenzo[a,h]anthracene (DBADE) and the fjord region diolepoxides ()-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]-pyrene (DBPDE) and (+/-)-anti-3,4-dihydroxy-1,2-epoxy-1,2,3,4-tetrahydrobenzo[c]-phenanthrene (BPhDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. The (32)P-postlabelling assay was applied to analyze DNA adduct levels and the Hprt gene mutation assay for monitoring mutations. Previously, we found that the mutagenicity per adduct was four times higher for DBPDE compared to BPDE in NER proficient cells. In these same cells, the mutagenicity of DBADE and BPhDE adducts was now found to be significantly lower compared to that of BPDE. In NER deficient cells the highest mutagenicity per adduct was found for BPDE and there was a tenfold and fivefold difference when comparing the BPDE data with the DBADE and BPhDE data, respectively. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the Hprt gene. Since NER turned out to be an important pathway for the yield of mutations, we further analyzed the role of transcription coupled NER versus global genome NER. However, our data demonstrate that neither of these pathways seems to be the sole factor determining the mutation frequency of the four PAH-DE and that the differences in the repair efficiency of these compounds could not be related to the presence of a bay or fjord region in the parent PAH.
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| 6. |
- McDonald, Karin R, et al.
(författare)
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The Pif1 family helicase Pfh1 facilitates telomere replication and has an RPA-dependent role during telomere lengthening
- 2014
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 24, s. 80-86
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Tidskriftsartikel (refereegranskat)abstract
- Pif1 family helicases are evolutionary conserved 5'-3' DNA helicases. Pfh1, the sole Schizosaccharomyces pombe Pif1 family DNA helicase, is essential for maintenance of both nuclear and mitochondrial DNAs. Here we show that its nuclear functions include roles in telomere replication and telomerase action. Pfh1 promoted semi-conservative replication through telomeric DNA, as replication forks moved more slowly through telomeres when Pfh1 levels were reduced. Unlike other organisms, S. pombe cells overexpressing Pfh1 displayed markedly longer telomeres. Because this lengthening occurred in the absence of homologous recombination but not in a replication protein A mutant (rad11-D223Y) that has defects in telomerase function, it is probably telomerase-mediated. The effects of Pfh1 on telomere replication and telomere length are likely direct as Pfh1 exhibited high telomere binding in cells expressing endogenous levels of Pfh1. These findings argue that Pfh1 is a positive regulator of telomere length and telomere replication.
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| 7. |
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| 8. |
- Parsons, Jason L., et al.
(författare)
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XRCC1 phosphorylation by CK2 is required for its stability and efficient DNA repair
- 2010
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Ingår i: DNA Repair. - : Elsevier BV. - 1568-7864 .- 1568-7856. ; 9:7, s. 835-841
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Tidskriftsartikel (refereegranskat)abstract
- XRCC1 is a scaffold protein that interacts with several DNA repair proteins and plays a critical role in DNA base excision repair (BER). XRCC1 protein is in a tight complex with DNA ligase III alpha (Lig III) and this complex is involved in the ligation step of both BER and repair of DNA single strand breaks. The majority of XRCC1 has previously been demonstrated to exist in a phosphorylated form and cells containing mutant XRCC1, that is unable to be phosphorylated, display a reduced rate of single strand break repair. Here, in an unbiased assay, we demonstrate that the cytoplasmic form of the casein kinase 2 (CK2) protein is the major protein kinase activity involved in phosphorylation of XRCC1 in human cell extracts and that XRCC1 phosphorylation is required for XRCC1-Lig III complex stability. We demonstrate that XRCC1-Lig III complex containing mutant XRCC1, in which CK2 phosphorylation sites have been mutated, is unstable. We also find that a knockdown of CK2 by siRNA results in both reduced XRCC1 phosphorylation and stability, which also leads to a reduced amount of Lig III and accumulation of DNA strand breaks. We therefore propose that CK2 plays an important role in DNA repair by contributing to the stability of XRCC1-Lig III complex.
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| 9. |
- Persson, Örjan, 1974, et al.
(författare)
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UspB, a member of the sigma-S regulon, facilitates RuvC resolvase function.
- 2010
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Ingår i: DNA repair. - : Elsevier BV. - 1568-7856 .- 1568-7864. ; 9:11, s. 1162-9
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Tidskriftsartikel (refereegranskat)abstract
- A growing body of evidence shows that there is an intimate connection between proteins required for genome stability and stationary phase survival. In this work we show that the integral membrane protein UspB, a member of the RpoS regulon, is required for proper DNA repair as mutants lacking uspB are hypersensitive to several DNA damaging agents including ultraviolet light, mitomycin C, bleomycin and ciprofloxacin. Genetic and physical studies demonstrate that UspB acts in the RuvABC recombination repair pathway and removing uspB creates a phenocopy of the Holliday junction resolvase mutant, ruvC. Further, we show that the uspB mutant phenotype can be suppressed by ectopic overproduction of RuvC and that both ruvC and uspB mutants can be suppressed by inactivating recD. The fact that RuvABC-dependent repair requires UspB for proper activity suggests that the sigma-S regulon works together with DNA repair pathways under stress conditions to defend the cell against genotoxic stress.
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| 10. |
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