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1.
Saccharomyces cerevisiae RAD5-encoded DNA repair protein contains DNA helicase and zinc-binding sequence motifs and affects the stability of simple repetitive sequences in the genome. 总被引:4,自引:0,他引:4
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R E Johnson S T Henderson T D Petes S Prakash M Bankmann L Prakash 《Molecular and cellular biology》1992,12(9):3807-3818
rad5 (rev2) mutants of Saccharomyces cerevisiae are sensitive to UV light and other DNA-damaging agents, and RAD5 is in the RAD6 epistasis group of DNA repair genes. To unambiguously define the function of RAD5, we have cloned the RAD5 gene, determined the effects of the rad5 deletion mutation on DNA repair, DNA damage-induced mutagenesis, and other cellular processes, and analyzed the sequence of RAD5-encoded protein. Our genetic studies indicate that RAD5 functions primarily with RAD18 in error-free postreplication repair. We also show that RAD5 affects the rate of instability of poly(GT) repeat sequences. Genomic poly(GT) sequences normally change length at a rate of about 10(-4); this rate is approximately 10-fold lower in the rad5 deletion mutant than in the corresponding isogenic wild-type strain. RAD5 encodes a protein of 1,169 amino acids of M(r) 134,000, and it contains several interesting sequence motifs. All seven conserved domains found associated with DNA helicases are present in RAD5. RAD5 also contains a cysteine-rich sequence motif that resembles the corresponding sequences found in 11 other proteins, including those encoded by the DNA repair gene RAD18 and the RAG1 gene required for immunoglobin gene arrangement. A leucine zipper motif preceded by a basic region is also present in RAD5. The cysteine-rich region may coordinate the binding of zinc; this region and the basic segment might constitute distinct DNA-binding domains in RAD5. Possible roles of RAD5 putative ATPase/DNA helicase activity in DNA repair and in the maintenance of wild-type rates of instability of simple repetitive sequences are discussed. 相似文献
2.
Aspergillus nidulans uvsBATR and scaANBS1 genes show genetic interactions during recovery from replication stress and DNA damage
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Fagundes MR Semighini CP Malavazi I Savoldi M de Lima JF de Souza Goldman MH Harris SD Goldman GH 《Eukaryotic cell》2005,4(7):1239-1252
The ATM/ATR kinases and the Mre11 (Mre11-Rad50-Nbs1) protein complex are central players in the cellular DNA damage response. Here we characterize possible interactions between Aspergillus nidulans uvsB(ATR) and the Mre11 complex (scaA(NBS1)). We demonstrate that there is an epistatic relationship between uvsB(ATR), the homolog of the ATR/MEC1 gene, and scaA(NBS1), the homolog of the NBS1/XRS2 gene, for both repair and checkpoint functions and that correct ScaA(NBS1) expression during recovery from replication stress depends on uvsB(ATR). In addition, we also show that the formation of UvsC foci during recovery from replication stress is dependent on both uvsB(ATR) and scaA(NBS1) function. Furthermore, ScaA(NBS1) is also dependent on uvsB(ATR) for nuclear focus formation upon the induction of DNA double-strand breaks by phleomycin. Our results highlight the extensive genetic interactions between UvsB and the Mre11 complex that are required for S-phase progression and recovery from DNA damage. 相似文献
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Semidominant suppressors of Srs2 helicase mutations of Saccharomyces cerevisiae map in the RAD51 gene, whose sequence predicts a protein with similarities to procaryotic RecA proteins. 总被引:14,自引:4,他引:10
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Eleven suppressors of the radiation sensitivity of Saccharomyces cerevisiae diploids lacking the Srs2 helicase were analyzed and found to contain codominant mutations in the RAD51 gene known to be involved in recombinational repair and in genetic recombination. These mutant alleles confer an almost complete block in recombinational repair, as does deletion of RAD51, but heterozygous mutant alleles suppress the defects of srs2::LEU2 cells and are semidominant in Srs2+ cells. The results of this study are interpreted to mean that wild-type Rad51 protein binds to single-stranded DNA and that the semidominant mutations do not prevent this binding. The cloning and sequencing of RAD51 indicated that the gene encodes a predicted 400-amino-acid protein with a molecular mass of 43 kDa. Sequence comparisons revealed homologies to domains of Escherichia coli RecA protein predicted to be involved in DNA binding, ATP binding, and ATP hydrolysis. The expression of RAD51, measured with a RAD51-lacZ gene fusion, was found to be UV- and gamma-ray-inducible, with dose-dependent responses. 相似文献
5.
The cellular role of the DNA polymerase encoded by the Saccharomyces cerevisiae POL4 gene is unclear. We have used an epistasis analysis to investigate whether the proteins encoded by the POL4 and RAD27 genes participate in alternative, non-redundant subpathways of DNA base excision repair (BER). We constructed strains in which the genes were deleted singly or in combination and have examined their sensitivity to DNA damaging agents as well as spontaneous mutation frequency. The double deletion strain is no more sensitive to damaging agents and has no higher spontaneous mutation frequency than the most sensitive single mutant. These data indicate that the protein encoded by the POL4 gene does not participate in a non-redundant subpathway of base excision repair under these conditions. We discuss the implications of these results in light of the recent classification of the POL4 gene product as a member of the DNA polymerase lambda family. 相似文献
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F K Khasanov G V Savchenko E V Bashkirova V G Korolev W D Heyer V I Bashkirov 《Genetics》1999,152(4):1557-1572
A new DNA repair gene from Schizosaccharomyces pombe with homology to RecA was identified and characterized. Comparative analysis showed highest similarity to Saccharomyces cerevisiae Rad55p. rhp55(+) (rad homologue pombe 55) encodes a predicted 350-amino-acid protein with an M(r) of 38,000. The rhp55Delta mutant was highly sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and, to a lesser degree, UV. These phenotypes were enhanced at low temperatures, similar to deletions in the S. cerevisiae RAD55 and RAD57 genes. Many rhp55Delta cells were elongated with aberrant nuclei and an increased DNA content. The rhp55 mutant showed minor deficiencies in meiotic intra- and intergenic recombination. Sporulation efficiency and spore viability were significantly reduced. Double-mutant analysis showed that rhp55(+) acts in one DNA repair pathway with rhp51(+) and rhp54(+), homologs of the budding yeast RAD51 and RAD54 genes, respectively. However, rhp55(+) is in a different epistasis group for repair of UV-, MMS-, or gamma-ray-induced DNA damage than is rad22(+), a putative RAD52 homolog of fission yeast. The structural and functional similarity suggests that rhp55(+) is a homolog of the S. cerevisiae RAD55 gene and we propose that the functional diversification of RecA-like genes in budding yeast is evolutionarily conserved. 相似文献
8.
The link between 20S proteasome activity and post-replication DNA repair in Saccharomyces cerevisiae
We have shown previously that deletion of the Saccharomyces cerevisiae UMP1 gene encoding the 20S proteasome maturase causes sensitivity to UV radiation. In the current report, we have extended this finding to show that mutations specifically compromising chymotrypsin-like or trypsin-like activity of 20S proteasome peptidases also result in increased UV sensitivity. We have also established that mutations affecting proteasome activity, namely ump1Delta, pre2-K108R and pup1-T20A, result in spontaneous and UV-induced mutator phenotypes. To elucidate the origin of these DNA repair phenotypes of the proteasomal mutants, we performed epistasis analysis, with respect to UV sensitivity, using yeast strains with the UMP1 deletion in different DNA repair backgrounds. We show that UMP1 is not epistatic to RAD23 and RAD2, which are involved in the nucleotide excision repair (NER) pathway. Instead, our results indicate that UMP1 as well as PUP1 and PRE2 (encoding catalytic subunits of 20S proteasome) belong to an epistatic group of genes functioning in post-replication DNA repair (PRR) and are hypostatic to RAD18, which, in complex with RAD6, plays a central role in PRR. We also show that UMP1 is epistatic to REV3 and RAD30, although the relationship of UMP1 with these genes is different. 相似文献
9.
A new DNA repair gene from fission yeast Schizosaccharomyces pombe rlp1+ (RecA-like protein) has been identified. Rlp1 shows homology to RecA-like proteins, and is the third S. pombe Rad51 paralog besides Rhp55 and Rhp57. The new gene encodes a 363 aa protein with predicted Mr of 41,700 and has NTP-binding motif. The rlp1Delta mutant is sensitive to methyl methanesulfonate (MMS), ionizing radiation (IR), and camptothecin (CPT), although to a lesser extent than the deletion mutants of rhp55+ and rhp51+ genes. In contrast to other recombinational repair mutants, the rlp1Delta mutant does not exhibit sensitivity to UV light and mitomycin C (MMC). Mitotic recombination is moderately reduced in rlp1 mutant. Epistatic analysis of MMS and IR-sensitivity of rlp1Delta mutant indicates that rlp1+ acts in the recombinational pathway of double-strand break (DSB) repair together with rhp51+, rhp55+, and rad22+ genes. Yeast two-hybrid analysis suggests that Rlp1 may interact with Rhp57 protein. We propose that Rlp1 have an accessory role in repair of a subset of DNA damage induced by MMS and IR, and is required for the full extent of DNA recombination and cell survival under condition of a replication fork collapse. 相似文献
10.
Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here. 相似文献
11.
To identify new human proteins implicated in homologous recombination (HR), we set up 'a papillae assay' to screen a human cDNA library using the RS112 strain of Saccharomyces cerevisiae containing an intrachromosomal recombination substrate. We isolated 23 cDNAs, 11 coding for complete proteins and 12 for partially deleted proteins that increased HR when overexpressed in yeast. We characterized the effect induced by the overexpression of the complete human proteasome subunit beta 2, the partially deleted proteasome subunits alpha 3 and beta 8, the ribosomal protein L12, the brain abundant membrane signal protein (BASP1) and the human homologue to v-Ha-RAS (HRAS), which elevated HR by 2-6.5-fold over the control. We found that deletion of the RAD52 gene, which has a key role in most HR events, abolished the increase of HR induced by the proteasome subunits and HRAS; by contrast, the RAD52 deletion did not affect the high level of HR due to BASP1 and RPL12. This suggests that the proteins stimulated yeast HR via different mechanisms. Overexpression of the complete beta 2 human proteasome subunit or the partially deleted alpha 3 and beta 8 subunits increased methyl methanesulphonate (MMS) resistance much more in the rad52 Delta mutant than in the wild-type. Overexpression of RPL12 and BASP1 did not affect MMS resistance in both the wild-type and the rad52 Delta mutant, whereas HRAS decreased MMS resistance in the rad52 Delta mutant. The results indicate that these proteins may interfere with the pathway(s) involved in the repair of MMS-induced DNA damage. Finally, we provide further evidence that yeast is a helpful tool to identify human proteins that may have a regulatory role in HR. 相似文献
12.
Mutational inactivation of the Saccharomyces cerevisiae RAD4 gene in Escherichia coli. 总被引:3,自引:1,他引:2
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The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. When plasmids containing the wild-type gene were transformed into various Escherichia coli strains, transformation frequencies were drastically reduced. Most plasmids recovered from transformants showed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidence of deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, little or no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesis of the wild-type RAD4 allele before transformation of E. coli restored transformation to normal levels. Plasmids recovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNA fragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethal to E. coli and show that lethality can be prevented by inactivation of the gene before transformation. Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4 gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, by inference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2, rad4-3, and rad4-4 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition, overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. These observations suggest that the Rad4 protein is relatively insensitive to mutational inactivation. 相似文献
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We have investigated the sensitivity to DNA-damaging agents of a strain of Saccharomyces cerevisiae containing a deletion of the RAD27 gene. The mutant strain is sensitive to a number of alkylating agents that modify DNA at a variety of positions, including one that produces primarily phosphotriesters. In contrast, the mutant strain is not sensitive to the oxidizing agent hydrogen peroxide. The introduction of a plasmid containing the FEN-1 gene (the human ortholog of the RAD27 gene) can substantially complement the sensitivity to alkylating agents observed in the mutant strain. 相似文献
15.
Analysis of the essential and excision repair functions of the RAD3 gene of Saccharomyces cerevisiae by mutagenesis. 总被引:13,自引:1,他引:12
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The RAD3 gene of Saccharomyces cerevisiae, which is involved in excision repair of DNA and is essential for cell viability, was mutagenized by site-specific and random mutagenesis. Site-specific mutagenesis was targeted to two regions near the 5' and 3' ends of the coding region, selected on the basis of amino acid sequence homology with known nucleotide binding and with known specific DNA-binding proteins, respectively. Two mutations in the putative nucleotide-binding region and one in the putative DNA-binding region inactivate the excision repair function of the gene, but not the essential function. A gene encoding two tandem mutations in the putative DNA-binding region is defective in both excision repair and essential functions of RAD3. Seven plasmids were isolated following random mutagenesis with hydroxylamine. Mutations in six of these plasmids were identified by gap repair of mutant plasmids from the chromosome of strains with previously mapped rad3 mutations, followed by DNA sequencing. Three of these contain missense mutations which inactivate only the excision repair function. The other three carry nonsense mutations which inactivate both the excision repair and essential functions. Collectively our results indicate that the RAD3 excision repair function is more sensitive to inactivation than is the essential function. Overexpression of wild-type Rad3 protein and a number of rad3 mutant proteins did not affect the UV resistance of wild-type yeast cells. However, overexpression of Rad3-2 protein rendered wild-type cells partially UV sensitive, indicating that excess Rad3-2 protein is dominant to the wild-type form. These and other results suggest that Rad3-2 protein retains its affinity for damaged DNA or other substrates, but is not catalytically active in excision repair. 相似文献
16.
Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells 总被引:7,自引:0,他引:7
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Tateishi S Niwa H Miyazaki J Fujimoto S Inoue H Yamaizumi M 《Molecular and cellular biology》2003,23(2):474-481
In lower eukaryotes, Rad18 plays a crucial role in postreplication repair. Previously, we isolated a human homologue of RAD18 (hRAD18) and showed that human cells overexpressing hRad18 protein with a mutation in the RING finger motif are defective in postreplication repair. Here, we report the construction of RAD18-knockout mouse embryonic stem cells by gene targeting. These cells had almost the same growth rate as wild-type cells and manifested phenotypes similar to those of human cells expressing mutant Rad18 protein: hypersensitivity to multiple DNA damaging agents and a defect in postreplication repair. Mutation was not induced in the knockout cells with any higher frequencies than in wild-type cells, as shown by ouabain resistance. In the knockout cells, spontaneous sister chromatid exchange (SCE) occurred with twice the frequency observed in normal cells. After mild DNA damage, SCE was threefold higher in the knockout cells, while no increase was observed in normal cells. Stable transformation efficiencies were approximately 20-fold higher in knockout cells, and gene targeting occurred with approximately 40-fold-higher frequency than in wild-type cells at the Oct3/4 locus. These results indicate that dysfunction of Rad18 greatly increases both the frequency of homologous as well as illegitimate recombination, and that RAD18 contributes to maintenance of genomic stability through postreplication repair. 相似文献
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p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction. 总被引:29,自引:1,他引:28
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H W Stürzbecher B Donzelmann W Henning U Knippschild S Buchhop 《The EMBO journal》1996,15(8):1992-2002
The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death. Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy. Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events. Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA. In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity. In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation. These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination. Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene. 相似文献
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Kawachi R Akashi T Kamitani Y Sy A Wangchaisoonthorn U Nihira T Yamada Y 《Molecular microbiology》2000,36(2):302-313