Specific interaction between DNA polymerase II (PolD) and RadB, a Rad51/Dmc1 homolog, in Pyrococcus furiosus.
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Pyrococcus furiosus has an operon containing the DNA polymerase II (PolD) gene and three other genes. Using a two-hybrid screening to examine the interactions of the proteins encoded by the operon, we identified a specific interaction between the second subunit of PolD (DP1) and a Rad51/Dmc1 homologous protein (RadB). To ensure the specific interaction between these two proteins, each gene in the operon was expressed in Escherichia coli or insect cells separately and the products were purified. The in vitro analyses using the purified proteins also showed the interaction between DP1 and RadB. The deletion mutant analysis of DP1 revealed that a region important for binding with RadB is located in the central part of the sequence (amino acid residues 206-498). This region has an overlap to the C-terminal half (amino acids 334-613), which is highly conserved among euryarchaeal DP1s and is essential for the activity of PolD. Our results suggest that, although RadB does not noticeably affect the primer extension ability of PolD in vitro, PolD may utilize the RadB protein in DNA synthesis under certain conditions. (+info)
Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells.
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Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways. Vertebrate Mre11 is essential. Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification. Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies. Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair. Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ. (+info)
A cell cycle-specific requirement for the XRCC1 BRCT II domain during mammalian DNA strand break repair.
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XRCC1 protein is essential for viability in mammals and is required for efficient DNA single-strand break repair and genetic stability following DNA base damage. We report here that XRCC1-dependent strand break repair in G(1) phase of the cell cycle is abolished by mutations created within the XRCC1 BRCT domain that interact with DNA ligase III. In contrast, XRCC1-dependent DNA strand break repair in S phase is largely unaffected by these mutations. These data describe a cell cycle-specific role for a BRCT domain, and we conclude that the XRCC1-DNA ligase III complex is required for DNA strand break repair in G(1) phase of the cell cycle but is dispensable for this process in S phase. The S-phase DNA repair process can remove both strand breaks induced in S phase and those that persist from G(1) and can in part compensate for lack of repair in G(1). This process correlates with the appearance of XRCC1 nuclear foci that colocalize with Rad51 and may thus function in concert with homologous recombination. (+info)
Efficient strand transfer by the RadA recombinase from the hyperthermophilic archaeon Desulfurococcus amylolyticus.
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The radA gene predicted to be responsible for homologous recombination in a hyperthermophilic archaeon, Desulfurococcus amylolyticus, was cloned, sequenced, and overexpressed in Escherichia coli cells. The deduced amino acid sequence of the gene product, RadA, was more similar to the human Rad51 protein (65% homology) than to the E. coli RecA protein (35%). A highly purified RadA protein was shown to exclusively catalyze single-stranded DNA-dependent ATP hydrolysis, which monitored presynaptic recombinational complex formation, at temperatures above 65 degrees C (catalytic rate constant of 1.2 to 2.5 min(-1) at 80 to 95 degrees C). The RadA protein alone efficiently promoted the strand exchange reaction at the range of temperatures from 80 to 90 degrees C, i.e., at temperatures approaching the melting point of DNA. It is noteworthy that both ATP hydrolysis and strand exchange are very efficient at temperatures optimal for host cell growth (90 to 92 degrees C). (+info)
Rapid exchange of A:T base pairs is essential for recognition of DNA homology by human Rad51 recombination protein.
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Human Rad51 belongs to a ubiquitous family of proteins that enable a single strand to recognize homology in duplex DNA, and thereby to initiate genetic exchanges and DNA repair, but the mechanism of recognition remains unknown. Kinetic analysis by fluorescence resonance energy transfer combined with the study of base substitutions and base mismatches reveals that recognition of homology, helix destabilization, exchange of base pairs, and initiation of strand exchange are integral parts of a rapid, concerted mechanism in which A:T base pairs play a critical role. Exchange of base pairs is essential for recognition of homology, and physical evidence indicates that such an exchange occurs early enough to mediate recognition. (+info)
c-Abl tyrosine kinase is not essential for ataxia telangiectasia mutated functions in chromosomal maintenance.
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c-Abl is activated by DNA damage in an ataxia telangiectasia mutated (ATM)-dependent manner and plays important roles in growth arrest and apoptosis induced by DNA damage. c-Abl also interacts physically and functionally with Rad51, a key molecule in homologous recombinational (HR) DNA repair. To study further the roles of c-Abl in HR DNA repair, we generated c-Abl(-/-) and ATM(-/-)/c-Abl(-/-) mutant cell lines from a chicken B lymphocyte DT40 cell line, comparing the phenotypes of these mutants to those of ATM(-/-) DT40 cells that we had created previously. We found that the time course of radiation-induced Rad51 focus formation is abnormal in ATM(-/-) DT40 cells, consistent with the observation that ATM(-/-) DT40 cells display hypersensitivity to ionizing radiation and highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In contrast, c-Abl(-/-) cells did not show these ATM-related defects in their cellular response to radiation, nor did the disruption of c-Abl in ATM(-/-) DT40 cells exacerbate these ATM-related defects. However, c-Abl(-/-) DT40 cells, but not ATM(-/-) DT40 cells, were resistant to radiation-induced apoptosis, indicating an important role for c-Abl in this cellular response to ionizing radiation. These results therefore indicate that, although ATM plays an important role in genome maintenance, c-Abl is not essential for this ATM function. These findings suggest that c-Abl and ATM play important roles in the maintenance of the cell homeostasis in response to DNA damage that are, at least in part, independent. (+info)
RAD51 is required for the repair of plasmid double-stranded DNA gaps from either plasmid or chromosomal templates.
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DNA double-strand breaks may be induced by endonucleases, ionizing radiation, chemical agents, and mechanical forces or by replication of single-stranded nicked chromosomes. Repair of double-strand breaks can occur by homologous recombination or by nonhomologous end joining. A system was developed to measure the efficiency of plasmid gap repair by homologous recombination using either chromosomal or plasmid templates. Gap repair was biased toward gene conversion events unassociated with crossing over using either donor sequence. The dependence of recombinational gap repair on genes belonging to the RAD52 epistasis group was tested in this system. RAD51, RAD52, RAD57, and RAD59 were required for efficient gap repair using either chromosomal or plasmid donors. No homologous recombination products were recovered from rad52 mutants, whereas a low level of repair occurred in the absence of RAD51, RAD57, or RAD59. These results suggest a minor pathway of strand invasion that is dependent on RAD52 but not on RAD51. The residual repair events in rad51 mutants were more frequently associated with crossing over than was observed in the wild-type strain, suggesting that the mechanisms for RAD51-dependent and RAD51-independent events are different. Plasmid gap repair was reduced synergistically in rad51 rad59 double mutants, indicating an important role for RAD59 in RAD51-independent repair. (+info)
Expression and nuclear localization of BLM, a chromosome stability protein mutated in Bloom's syndrome, suggest a role in recombination during meiotic prophase.
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Bloom's syndrome (BS) is a recessive human genetic disorder characterized by short stature, immunodeficiency and elevated risk of malignancy. BS cells have genomic instability and an increased frequency of sister chromatid exchange. The gene mutated in BS, BLM, encodes a 3'-5' helicase (BLM) with homology to bacterial recombination factor, RecQ. Human males homozygous for BLM mutations are infertile and heterozygous individuals display increased frequencies of structural chromosome abnormalities in their spermatozoa. Also, mutations in the Saccharomyces cerevisiae homolog of BLM, Sgs1, cause a delay in meiotic nuclear division and a reduction in spore viability. These observations suggest that BLM may play a role during meiosis. Our antibodies raised against the C terminus of the human protein specifically recognize both mouse and human BLM in western blots of cell lines and in successive developmental stages of spermatocytes, but fail to detect BLM protein in a cell line with a C-terminally truncated protein. BLM protein expression and location are detected by immunofluorescence and immunoelectron microscopy as discrete foci that are sparsely present on early meiotic prophase chromosome cores, later found abundantly on synapsed cores, frequently in combination with the recombinases RAD51 and DMC1, and eventually as pure BLM foci. The colocalization of RAD51/DMC1 with BLM and the statistically significant excess of BLM signals in the synapsed pseudoautosomal region of the X-Y chromosomes, which is a recombinational hot spot, provide indications that BLM protein may function in the meiotic recombination process. (+info)