Werner syndrome is an autosomal recessive disorder associated with premature aging and cancer predisposition caused by mutations of the WRN gene. WRN is a member of the RecQ DNA helicase family with functions in maintaining genome stability. Sir2, an NAD-dependent histone deacetylase, has been proven to extend life span in yeast and Caenorhabditis elegans. Mammalian Sir2 (SIRT1) has also been found to regulate premature cellular senescence induced by the tumor suppressors PML and p53. SIRT1 plays an important role in cell survival promoted by calorie restriction. Here we show that SIRT1 interacts with WRN both in vitro and in vivo; this interaction is enhanced after DNA damage. WRN can be acetylated by acetyltransferase CBP/p300, and SIRT1 can deacetylate WRN both in vitro and in vivo. WRN acetylation decreases its helicase and exonuclease activities, and SIRT1 can reverse this effect. WRN acetylation alters its nuclear distribution. Down-regulation of SIRT1 reduces WRN translocation from nucleoplasm to nucleoli after DNA damage. These results suggest that SIRT1 regulates WRN-mediated cellular responses to DNA damage through deacetylation of WRN. Werner syndrome (WS)3 is a human autosomal recessive disorder that displays symptoms of premature aging, including graying and loss of hair, wrinkling and ulceration of skin, atherosclerosis, osteoporosis, and cataracts. WS patients also exhibit an increased incidence of diabetes mellitus type 2, hypertension, and are highly disposed to cancers (1). WS results from mutation of the WRN gene, a member of the RecQ DNA helicase family (2). Mutations in other family members, BLM and RECQ4, are responsible for the two other cancer-prone and premature aging syndromes, Bloom (3) and Rothmund-Thomson (4), respectively. Consistent with other RecQ helicases, WRN protein possesses 3Ј to 5Ј DNA helicase activity; however, it is the only human RecQ member to also have a 3Ј to 5Ј exonuclease activity. Although its physiological substrate is not yet clear, WRN appears to preferentially act on replication and recombination structures. Cells from WS patients show premature replicative senescence compared with cells derived from normal individuals (5). WS cells also show hypersensitivity to selected DNA-damaging agents including 4-nitroquinoline-1-oxide (4NQO; 6), topoisomerase inhibitors (7), and certain DNA cross-linking agents (8). Compared with normal cells, WS cells also exhibit increased genomic instability including higher levels of DNA deletions, translocations, and chromosomal breaks (9, 10), suggesting that WRN plays an important role in one or more genome maintenance pathways (11).WRN protein shows dynamic relocalization within the nucleus under different conditions of growth. The WRN protein localizes to the nucleoli in a variety of cell types (12), and this localization is modulated by DNA damage and cell cycle. Upon serum starvation or treatment with hydroxyurea (HU), aphidicolin, 4NQO, etoposide, or camptothecin, WRN migrates from nucleoli to discrete nuclear foci (13-17). ...

BackgroundThe premature aging and cancer-prone Werner and Bloom syndromes are caused by defects in the RecQ helicase enzymes WRN and BLM, respectively. Recently, both WRN and BLM (as well as several other RecQ members) have been shown to possess a strand annealing activity in addition to the requisite DNA unwinding activity. Since an annealing function would appear to directly oppose the action of a helicase, we have examined in this study the dynamic equilibrium between unwinding and annealing mediated by either WRN or BLM.ResultsOur investigation into the competition between annealing and unwinding demonstrates that, under standard reaction conditions, WRN- or BLM-mediated annealing can partially or completely mask unwinding as measured in standard helicase assays. Several strategies were employed to suppress the annealing activity so that the actual strength of WRN- or BLM-dependent unwinding could be more accurately assessed. Interestingly, if a DNA oligomer complementary to one strand of the DNA substrate to be unwound is added during the helicase reaction, both WRN and BLM unwinding is enhanced, presumably by preventing protein-mediated re-annealing. This strategy allowed measurement of WRN-catalyzed unwinding of long (80 base pair) duplex regions and fully complementary, blunt-ended duplexes, both of which were otherwise quite refractory to the helicase activity of WRN. Similarly, the addition of trap strand stimulated the ability of BLM to unwind long and blunt-ended duplexes. The stimulatory effect of the human replication protein A (hRPA, the eukaryotic single-stranded DNA binding protein) on both WRN- and BLM-dependent unwinding was also re-examined in light of its possible role in preventing re-annealing. Our results show that hRPA influences the outcome of WRN and BLM helicase assays by both inhibiting re-annealing and directly promoting unwinding, with the larger contribution from the latter mechanism.ConclusionThese findings indicate that measurements of unwinding by WRN, BLM, and probably other RecQ helicases are complicated by their annealing properties. Thus, WRN- and BLM-dependent unwinding activities are significantly stronger than previously believed. Since this broadens the range of potential physiological substrates for WRN and BLM, our findings have relevance for understanding their functions in vitro and in vivo.

BackgroundWRN is a multi-functional protein involving DNA replication, recombination and repair. WRN acetylation has been demonstrated playing an important role in response to DNA damage. We previously found that WRN acetylation can regulate its enzymatic activities and nuclear distribution.Methodology/Principal FindingHere, we investigated the factors involved in WRN acetylation and found that CBP and p300 are the only major acetyltransferases for WRN acetylation. We further identified 6 lysine residues in WRN that are subject to acetylation. Interestingly, WRN acetylation can increase its protein stability. SIRT1-mediated deacetylation of WRN reverses this effect. CBP dramatically increases the half-life of wild type WRN, while mutation of these 6 lysine residues (WRN-6KR) abrogates this increase. We further found that WRN stability is regulated by the ubiquitination pathway and WRN acetylation by CBP significantly reduces its ubiquitination. Importantly, we found that WRN is strongly acetylated and stabilized in response to mitomycin C (MMC) treatment. H1299 cells stably expressing WRN-6KR, which mimics unacetylated WRN, display significantly higher MMC sensitivity compared with the cells expressing wild-type WRN.Conclusion/SignificanceTaken together, these data demonstrate that WRN acetylation regulates its stability and has significant implications regarding the role of acetylation on WRN function in response to DNA damage.

The premature aging and cancer-prone disease Werner syndrome is caused by loss of function of the RecQ helicase family member Werner syndrome protein (WRN). At the cellular level, loss of WRN results in replication abnormalities and chromosomal aberrations, indicating that WRN plays a role in maintenance of genome stability. Consistent with this notion, WRN possesses annealing, exonuclease, and ATPase-dependent helicase activity on DNA substrates, with particularly high affinity for and activity on replication and recombination structures. After certain DNA-damaging treatments, WRN is recruited to sites of blocked replication and co-localizes with the human single-stranded DNA-binding protein replication protein A (RPA). In this study we examined the physical and functional interaction between WRN and RPA specifically in relation to replication fork blockage. Co-immunoprecipitation experiments demonstrated that damaging treatments that block DNA replication substantially increased association between WRN and RPA in vivo, and a direct interaction between purified WRN and RPA was confirmed. Furthermore, we examined the combined action of RPA (unmodified and hyperphosphorylation mimetic) and WRN on model replication fork and gapped duplex substrates designed to bind RPA. Even with RPA bound stoichiometrically to this gap, WRN efficiently catalyzed regression of the fork substrate. Further analysis showed that RPA could be displaced from both substrates by WRN. RPA displacement by WRN was independent of its ATPase- and helicase-dependent remodeling of the fork. Taken together, our results suggest that, upon replication blockage, WRN and RPA functionally interact and cooperate to help properly resolve replication forks and maintain genome stability.

The dual specificity phosphatase Cdc25A is a key regulator of the cell cycle that promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. In response to genotoxicants, Cdc25A undergoes posttranslational modifications which contribute to its proteasome-mediated degradation and consequent cell cycle checkpoint arrest. The most thoroughly studied Cdc25A modification is phosphorylation. We now provide the first evidence that Cdc25A can be acetylated and that it directly interacts with the ARD1 acetyltransferase which acetylates Cdc25A both biochemically and in cultured cells. When acetylated, Cdc25A has an extended half-life. We have also identified the class IV histone deacetylase, HDAC11, as a Cdc25A deacetylase. We further show that DNA damage, such as exposure to methyl methanesulfonate (MMS), etoposide or arsenic, increases Cdc25A acetylation. Importantly, this acetylation modulates Cdc25A phosphatase activity and its function as a cell cycle regulator, and may reflect a cellular response to DNA damage. Since Cdc25A, ARD1, and HDAC11 are frequently dysregulated in multiple types of cancer, our findings may provide insight into a novel mechanism in carcinogenesis.