Werner's syndrome (WS) is a rare autosomal recessive disorder that arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. The cellular function of WRN is still unclear, but on the basis of the cellular phenotypes of WS and of RecQ yeast mutants, its possible role in controlling recombination and/or in maintenance of genomic integrity during S-phase has been envisaged. With the use of two drugs, camptothecin and hydroxyurea, which produce replication-associated DNA damage and/or inhibit replication fork progression, we find that WS cells have a slower rate of repair associated with DNA damage induced in the S-phase and a reduced induction of RAD51 foci. As a consequence, WS cells undergo apoptotic cell death more than normal cells, even if they arrest and resume DNA synthesis at an apparently normal rate. Furthermore, we report that WS cells show a higher background level of DNA strand breaks and an elevated spontaneous induction of RAD51 foci. Our findings support the hypothesis that WRN could be involved in the correct resolution of recombinational intermediates that arise from replication arrest due to either DNA damage or replication fork collapse. INTRODUCTIONWerner's syndrome (WS) is a rare autosomal recessive disorder characterized by premature aging (Salk et al., 1985) and early onset of various neoplasms, including different types of carcinomas and sarcomas (Goto et al., 1981;Hrabko et al., 1982;Sato et al., 1988). This disorder arises as a consequence of mutations in a gene coding for a protein that is a member of RecQ family of DNA helicases, WRN. One of the hallmarks of WS patients is the genomic instability as evidenced by the spontaneous chromosome anomalies and large deletions in many genes (Salk et al., 1985;Fukuchi et al., 1989). It has been demonstrated that WRN exhibits DNA unwinding activity (Gray et al., 1997;Suzuki et al., 1997) and exonuclease activity residing in the N-terminal region (Huang et al., 1998). The cellular function of WRN is still unclear. It has been proposed that helicases are required for various DNA metabolic activities, including progression of replication fork, segregation of newly replicated chromosomes, disruption of the nucleosome structure, DNA supercoiling, transcription, recombination, and repair (Duguet, 1997). In addition, it has been suggested that RecQ family of DNA helicases has an important role in the maintenance of genomic integrity during DNA replication . Studies from yeast show that DNA replication does not proceed normally in absence of RecQ helicase function (Stewart et al., 1997) and in Xenopus laevis the ortholog of WRN is absolutely required for proper formation of replication foci (Yan et al., 1998). Functional interaction between proteins involved in DNA replication, such as replication protein A (RPA) and DNA polymerase ␦, and WRN also have been reported (Shen et al., 1998;Brosh et al., 1999;Kamath-Loeb et al., 2000). Furthermore, in yeast, the RecQlike proteins seem involved in ...

Cockayne syndrome (CS) is a rare genetic disease characterized by neurological problems, growth failure and premature ageing. Many of these features cannot simply be ascribed to the defect that CS cells display during transcription-coupled repair. Here, we show that CSB mutant cells are unable to react to hypoxic stimuli by properly activating the hypoxia-inducible factor-1 (HIF-1) pathway, a defect that is further enhanced in the event of a concomitant genotoxic stress. Furthermore, we show that CSB expression is under the control of HIF-1 and has a critical function during hypoxic response by redistributing p300 between HIF-1 and p53. Altogether, our data demonstrate that CSB is part of a feedback loop mechanism that modulates the biological functions of p53. The outcome of this study provides new insights into the understanding of the molecular basis of the CS phenotype and the involvement of the CSB protein in the hypoxic response pathway.

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In yeast, MSH2 plays an important role in mismatch repair (MMR) and recombination, whereas the function of the mammalian MSH2 protein in recombinational repair is not completely established. We examined the cellular responses of MSH2-deficient mouse cells to X-rays to clarify the role of MSH2 in recombinational repair. Cell survival, checkpoint functions and relocalization of the recombination-related proteins MRE11 and RAD51 were analysed in embryonic fibroblasts derived from MSH2 +/+ and MSH2 À/À mice, and in MSH2-proficient and deficient mouse colorectal carcinoma cells. Loss of MSH2 function was found to be associated with reduction in cell survival following radiation, absence of either MRE11 or RAD51 relocalization and a higher level of X-ray-induced chromosomal damage specifically in G2-phase cells. Finally, MSH2 À/À cells showed an inefficient early G2/M checkpoint, being arrested only transiently after irradiation before progressing into mitosis. Consistent with the premature release from the G2-phase arrest, activation of CHK1 was transient and CHK2 was not phosphorylated in synchronized MSH2-null cells. Our data suggest that an active MSH2 is required for a correct response to ionizing radiation-induced DNA damage in the G2 phase of the cell cycle, possibly connecting DSB repair to checkpoint signalling.