Redundancy of DNA helicases in p53-mediated apoptosis.
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A subset of DNA helicases, the RecQ family, has been found to be associated with the p53-mediated apoptotic pathway and is involved in maintaining genomic integrity. This family contains the BLM and WRN helicases, in which germline mutations are responsible for Bloom and Werner syndromes, respectively. TFIIH DNA helicases, XPB and XPD, are also components in this apoptotic pathway. We hypothesized that there may be some redundancy between helicases in their ability to complement the attenuated p53-mediated apoptotic levels seen in cells from individuals with diseases associated with these defective helicase genes. The attenuated apoptotic phenotype in Bloom syndrome cells was rescued not only by ectopic expression of BLM, but also by WRN or XPB, both 3' --> 5' helicases, but not expression of the 5' --> 3' helicase XPD. Overexpression of Sgs1, a WRN/BLM yeast homolog, corrected the reduction in BS cells only, which is consistent with Sgs1 being evolutionarily most homologous to BLM. A restoration of apoptotic levels in cells from WS, XPB or XPD patients was attained only by overexpression of the specific helicase. Our data suggest a limited redundancy in the pathways of these RecQ helicases in p53-induced apoptosis. (+info)
Competition between the DNA unwinding and strand pairing activities of the Werner and Bloom syndrome proteins.
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BACKGROUND: The 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. RESULTS: Our 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. CONCLUSION: These 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. (+info)
Interaction of Werner and Bloom syndrome genes with p53 in familial breast cancer.
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Mutations of the human RecQ helicase genes WRN and BLM lead to rare autosomal recessive disorders, Werner and Bloom syndromes, which are associated with premature ageing and cancer predisposition. We tested the hypothesis whether three polymorphic, non-conservative amino acid exchanges in WRN and BLM act as low-penetrance familial breast cancer risk factors. Moreover, we examined the putative impact of p53 MspI 1798G>A, which is completely linked to p53PIN3, a 16 bp insertion/duplication that has been associated with reduced p53 expression, on familial breast cancer risk. Genotyping analyses, performed on 816 BRCA1/2 mutation-negative German familial breast cancer patients and 1012 German controls, revealed a significant association of the WRN Cys1367Arg polymorphism with familial breast cancer (OR = 1.28, 95% CI 1.06-1.54) and high-risk familial breast cancer (OR = 1.32, 95% CI 1.06-1.65). The analysis of p53 MspI 1798G>A, which is completely linked to p53PIN3, showed a significantly increased familial breast cancer risk for carriers of the 16 bp insertion/duplication, following a recessive mode (OR = 2.15, 95% CI = 1.12-4.11). WRN Cys1367Arg, located in the C-terminus, the binding site of p53, is predicted to be damaging. The joint effect of WRN Cys1367Arg and p53 MspI resulted in an increased breast cancer risk compared to the single polymorphisms (OR = 3.39, 95% CI 1.19-9.71). In conclusion, our study indicates the importance of inherited variants in the WRN and p53 genes for familial breast cancer susceptibility. (+info)
BLAP75/RMI1 promotes the BLM-dependent dissolution of homologous recombination intermediates.
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BLM encodes a member of the highly conserved RecQ DNA helicase family, which is essential for the maintenance of genome stability. Homozygous inactivation of BLM gives rise to the cancer predisposition disorder Bloom's syndrome. A common feature of many RecQ helicase mutants is a hyperrecombination phenotype. In Bloom's syndrome, this phenotype manifests as an elevated frequency of sister chromatid exchanges and interhomologue recombination. We have shown previously that BLM, together with its evolutionarily conserved binding partner topoisomerase IIIalpha (hTOPO IIIalpha), can process recombination intermediates that contain double Holliday junctions into noncrossover products by a mechanism termed dissolution. Here we show that a recently identified third component of the human BLM/hTOPO IIIalpha complex, BLAP75/RMI1, promotes dissolution catalyzed by hTOPO IIIalpha. This activity of BLAP75/RMI1 is specific for dissolution catalyzed by hTOPO IIIalpha because it has no effect in reactions containing either Escherichia coli Top1 or Top3, both of which can also catalyze dissolution in a BLM-dependent manner. We present evidence that BLAP75/RMI1 acts by recruiting hTOPO IIIalpha to double Holliday junctions. Implications of the conserved ability of type IA topoisomerases to catalyze dissolution and how the evolution of factors such as BLAP75/RMI1 might confer specificity on the execution of this process are discussed. (+info)
Control of translocations between highly diverged genes by Sgs1, the Saccharomyces cerevisiae homolog of the Bloom's syndrome protein.
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Sgs1 is a RecQ family DNA helicase required for genome stability in Saccharomyces cerevisiae whose human homologs BLM, WRN, and RECQL4 are mutated in Bloom's, Werner, and Rothmund Thomson syndromes, respectively. Sgs1 and mismatch repair (MMR) are inhibitors of recombination between similar but divergent (homeologous) DNA sequences. Here we show that SGS1, but not MMR, is critical for suppressing spontaneous, recurring translocations between diverged genes in cells with mutations in the genes encoding the checkpoint proteins Mec3, Rad24, Rad9, or Rfc5, the chromatin assembly factors Cac1 or Asf1, and the DNA helicase Rrm3. The S-phase checkpoint kinase and telomere maintenance factor Tel1, a homolog of the human ataxia telangiectasia (ATM) protein, prevents these translocations, whereas the checkpoint kinase Mec1, a homolog of the human ATM-related protein, and the Rad53 checkpoint kinase are not required. The translocation structures observed suggest involvement of a dicentric intermediate and break-induced replication with multiple cycles of DNA template switching. (+info)
Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice.
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Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans. (+info)
MPS1-dependent mitotic BLM phosphorylation is important for chromosome stability.
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Spindle assembly checkpoint (SAC) ensures bipolar attachment of chromosomes to the mitotic spindle and is essential for faithful chromosome segregation, thereby preventing chromosome instability (CIN). Genetic evidence suggests a causal link between compromised SAC, CIN, and cancer. Bloom syndrome (BS) is a genetic disorder that predisposes affected individuals to cancer. BS cells exhibit elevated rates of sister chromatid exchange, chromosome breaks, and CIN. The BS gene product, BLM, is a member of the RecQ helicases that are required for maintenance of genome stability. The BLM helicase interacts with proteins involved in DNA replication, recombination, and repair and is required for the repair of stalled-replication forks and in the DNA damage response. Here we present biochemical evidence to suggest a role of BLM phosphorylation during mitosis in maintaining chromosome stability. BLM is associated with the SAC kinase MPS1 and is phosphorylated at S144 in a MPS1-dependent manner. Phosphorylated BLM interacts with polo-like kinase 1, a mitotic kinase that binds to phosphoserine/threonine through its polo-box domain (PBD). Furthermore, BS cells expressing BLM-S144A show normal levels of sister chromatid exchange but fail to maintain the mitotic arrest when SAC is activated and exhibit a broad distribution of chromosome numbers. We propose that MPS1-dependent BLM phosphorylation is important for ensuring accurate chromosome segregation, and its deregulation may contribute to cancer. (+info)
Sit down, relax and unwind: structural insights into RecQ helicase mechanisms.
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Helicases are specialized molecular motors that separate duplex nucleic acids into single strands. The RecQ family of helicases functions at the interface of DNA replication, recombination and repair in bacterial and eukaryotic cells. They are key, multifunctional enzymes that have been linked to three human diseases: Bloom's, Werner's and Rothmund-Thomson's syndromes. This review summarizes recent studies that relate the structures of RecQ proteins to their biochemical activities. (+info)