A common regulatory region shared by divergently transcribed genes of the Escherichia coli SOS system

The Escherichia coli single-stranded DNA binding protein (SSB) is implicated in DNA replication, recombination and repair. On the chromosome, the ssb gene is located adjacent to the excision repair gene uvrA, but the two genes are transcribed in opposite directions. uvrA has been shown to be part of the E. coli SOS system by introducing Mud(Ap, lac) insertions distal to the regulatory region of the gene in the chromosome. Recent investigations suggest that SSB is also involved in the SOS response. However, because the SSB protein is essential to the cell, the inducibility of the ssb gene cannot be investigated by the insertion method. Therefore, we used plasmids harbouring the regulatory region of ssb fused to the galK structural gene, while leaving an intact ssb gene in the chromosome. We show here that expression of the ssb gene is dependent on two promoters of which one is damage inducible. Evidence is presented that the divergently transcribed ssb and uvrA genes are controlled by a common LexA binding site.     

A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers

Cyclin D1 is a component of the core cell cycle machinery. Abnormally high levels of cyclin D1 are detected in many human cancer types. To elucidate the molecular functions of cyclin D1 in human cancers, we performed a proteomic screen for cyclin D1 protein partners in several types of human tumours. Analyses of cyclin D1 interactors revealed a network of DNA repair proteins, including RAD51, a recombinase that drives the homologous recombination process. We found that cyclin D1 directly binds RAD51, and that cyclin D1-RAD51 interaction is induced by radiation. Like RAD51, cyclin D1 is recruited to DNA damage sites in a BRCA2-dependent fashion. Reduction of cyclin D1 levels in human cancer cells impaired recruitment of RAD51 to damaged DNA, impeded the homologous recombination-mediated DNA repair, and increased sensitivity of cells to radiation in vitro and in vivo. This effect was seen in cancer cells lacking the retinoblastoma protein, which do not require D-cyclins for proliferation. These findings reveal an unexpected function of a core cell cycle protein in DNA repair and suggest that targeting cyclin D1 may be beneficial also in retinoblastoma-negative cancers which are currently thought to be unaffected by cyclin D1 inhibition.     

A ribonucleotide reductase gene involved in a p53-dependent cell-cycle checkpoint for DNA damage

The p53 gene is frequently inactivated in human cancers. Here we have isolated a p53-inducible gene, p53R2, by using differential display to examine messenger RNAs in a cancer-derived human cell line carrying a highly regulated wild-type p53 expression system. p53R2 contains a p53-binding sequence in intron 1 and encodes a 351-amino-acid peptide with striking similarity to the ribonucleotide reductase small subunit (R2), which is important in DNA synthesis during cell division. Expression of p53R2, but not R2, was induced by ultraviolet and gamma-irradiation and adriamycin treatment in a wild-type p53-dependent manner. Induction of p53R2 in p53-deficient cells caused G2/M arrest and prevented cells from death in response to adriamycin. Inhibition of endogenous p53R2 expression in cells that have an intact p53-dependent DNA damage checkpoint reduced ribonucleotide reductase activity, DNA repair and cell survival after exposure to various genotoxins. Our results indicate that p53R2 encodes a ribonucleotide reductase that is directly involved in the p53 checkpoint for repair of damaged DNA. The discovery of p53R2 clarifies a relationship between a ribonucleotide reductase activity involved in repair of damaged DNA and tumour suppression by p53.     

The lyase activity of the DNA repair protein beta-polymerase protects from DNA-damage-induced cytotoxicity

Small DNA lesions such as oxidized or alkylated bases are repaired by the base excision repair (BER) pathway. BER includes removal of the damaged base by a lesion-specific DNA glycosylase, strand scission by apurinic/apyrimidinic endonuclease, DNA resynthesis and ligation. BER may be further subdivided into DNA beta-polymerase (beta-pol)-dependent single-nucleotide repair and beta-pol-dependent or -independent long patch repair subpathways. Two important enzymatic steps in mammalian single-nucleotide BER are contributed by beta-pol: DNA resynthesis of the repair patch and lyase removal of 5'-deoxyribose phosphate (dRP). Fibroblasts from beta-pol null mice are hypersensitive to mono-functional DNA-methylating agents, resulting in increases in chromosomal damage, apoptosis and necrotic cell death. Here we show that only the dRP lyase activity of beta-pol is required to reverse methylating agent hypersensitivity in beta-pol null cells. These results indicate that removal of the dRP group is a pivotal step in BER in vivo. Persistence of the dRP moiety in DNA results in the hypersensitivity phenotype of beta-pol null cells and may signal downstream events such as apoptosis and necrotic cell death.     

DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1

A single double-strand break (DSB) induced by HO endonuclease triggers both repair by homologous recombination and activation of the Mec1-dependent DNA damage checkpoint in budding yeast. Here we report that DNA damage checkpoint activation by a DSB requires the cyclin-dependent kinase CDK1 (Cdc28) in budding yeast. CDK1 is also required for DSB-induced homologous recombination at any cell cycle stage. Inhibition of homologous recombination by using an analogue-sensitive CDK1 protein results in a compensatory increase in non-homologous end joining. CDK1 is required for efficient 5' to 3' resection of DSB ends and for the recruitment of both the single-stranded DNA-binding complex, RPA, and the Rad51 recombination protein. In contrast, Mre11 protein, part of the MRX complex, accumulates at unresected DSB ends. CDK1 is not required when the DNA damage checkpoint is initiated by lesions that are processed by nucleotide excision repair. Maintenance of the DSB-induced checkpoint requires continuing CDK1 activity that ensures continuing end resection. CDK1 is also important for a later step in homologous recombination, after strand invasion and before the initiation of new DNA synthesis.     

DNA-bound structures and mutants reveal abasic DNA binding by APE1 and DNA repair coordination [corrected

Non-coding apurinic/apyrimidinic (AP) sites in DNA are continually created in cells both spontaneously and by damage-specific DNA glycosylases. The biologically critical human base excision repair enzyme APE1 cleaves the DNA sugar-phosphate backbone at a position 5' of AP sites to prime DNA repair synthesis. Here we report three co-crystal structures of human APE1 bound to abasic DNA which show that APE1 uses a rigid, pre-formed, positively charged surface to kink the DNA helix and engulf the AP-DNA strand. APE1 inserts loops into both the DNA major and minor grooves and binds a flipped-out AP site in a pocket that excludes DNA bases and racemized beta-anomer AP sites. Both the APE1 active-site geometry and a complex with cleaved AP-DNA and Mn2+ support a testable structure-based catalytic mechanism. Alanine substitutions of the residues that penetrate the DNA helix unexpectedly show that human APE1 is structurally optimized to retain the cleaved DNA product. These structural and mutational results show how APE1 probably displaces bound glycosylases and retains the nicked DNA product, suggesting that APE1 acts in vivo to coordinate the orderly transfer of unstable DNA damage intermediates between the excision and synthesis steps of DNA repair.     

Molecular basis of xeroderma pigmentosum group C DNA recognition by engineered meganucleases

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers. The use of rare cutting DNA endonucleases-such as homing endonucleases, also known as meganucleases-constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules-Amel3-Amel4 and Ini3-Ini4-cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.     

Renaturation of DNA catalysed by yeast DNA repair and recombination protein RAD10.

The RAD10 gene of Saccharomyces cerevisiae is required for the incision step of excision repair of ultraviolet-damaged DNA, and it functions in mitotic recombination. RAD10 has homology to the human excision repair gene ERCC-1. Here we describe the purification of the protein encoded by RAD10 and show that it is a DNA-binding protein with a strong preference for single-stranded DNA. We also show that RAD10 promotes the renaturation of complementary DNA strands.     

Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins.

Xeroderma pigmentosum (XP), a human autosomal recessive disorder, is characterized by extreme sensitivity to sunlight and high incidence of skin cancers. XP cells are defective in the incision step of excision repair of DNA damaged by ultraviolet light. Cell fusion studies have defined seven XP complementation groups, XP-A to XP-G. Similar genetic complexity of excision repair is observed in the yeast Saccharomyces cerevisiae. Mutations in any one of five yeast genes, RAD1, RAD2, RAD3, RAD4, and RAD10, cause a total defect in incision and an extreme sensitivity to ultraviolet light. Here we report the characterization of the yeast RAD14 gene. The available rad14 point mutant is only moderately ultraviolet-sensitive, and it performs a substantial amount of incision of damaged DNA. Our studies with the rad14 deletion (delta) mutation indicate an absolute requirement of RAD14 in incision. RAD14 encodes a highly hydrophilic protein of 247 amino acids containing zinc-finger motifs, and it is similar to the protein encoded by the human XPAC gene that complements XP group A cell lines.     

E. coli recA protein-directed cleavage of phage lambda repressor requires polynucleotide

The recA protein mediates both genetic recombination and several cellular responses to DNA damage, including the induction of temperate bacteriophage. Indication of phage lambda results from proteolytic cleavage of lambda repressor directed by recA protein. We show here that this cleavage reaction requires both polynucleotide and ATP. We suggest that a stoichiometric complex of recA protein and DNA is active both to destroy repressors by proteolytic cleavage and to initiate pairing of this DNA to its homologous sequence in a DNA duplex ('strand invasion').     

Functional link of BRCA1 and ataxia telangiectasia gene product in DNA damage response

BRCA1 encodes a familial breast cancer suppressor that has a critical role in cellular responses to DNA damage. Mouse cells deficient for Brca1 show genetic instability, defective G2-M checkpoint control and reduced homologous recombination. BRCA1 also directly interacts with proteins of the DNA repair machinery and regulates expression of both the p21 and GADD45 genes. However, it remains unclear how DNA damage signals are transmitted to modulate the repair function of BRCA1. Here we show that the BRCA1-associated protein CtIP becomes hyperphosphorylated and dissociated from BRCA1 upon ionizing radiation. This phosphorylation event requires the protein kinase (ATM) that is mutated in the disease ataxia telangiectasia. ATM phosphorylates CtIP at serine residues 664 and 745, and mutation of these sites to alanine abrogates the dissociation of BRCA1 from CtIP, resulting in persistent repression of BRCA1-dependent induction of GADD45 upon ionizing radiation. We conclude that ATM, by phosphorylating CtIP upon ionizing radiation, may modulate BRCA1-mediated regulation of the DNA damage-response GADD45 gene, thus providing a potential link between ATM deficiency and breast cancer.     

Targeting the DNA repair defect in BRCA mutant cells as a therapeutic strategy

BRCA1 and BRCA2 are important for DNA double-strand break repair by homologous recombination, and mutations in these genes predispose to breast and other cancers. Poly(ADP-ribose) polymerase (PARP) is an enzyme involved in base excision repair, a key pathway in the repair of DNA single-strand breaks. We show here that BRCA1 or BRCA2 dysfunction unexpectedly and profoundly sensitizes cells to the inhibition of PARP enzymatic activity, resulting in chromosomal instability, cell cycle arrest and subsequent apoptosis. This seems to be because the inhibition of PARP leads to the persistence of DNA lesions normally repaired by homologous recombination. These results illustrate how different pathways cooperate to repair damage, and suggest that the targeted inhibition of particular DNA repair pathways may allow the design of specific and less toxic therapies for cancer.     

锯缘青蟹精氨酸激酶基因的克隆与表达

通过分子生物学方法,克隆得到锯缘青蟹（Scylla serrata）精氨酸激酶（Arginine Kinase,AK）开放阅读框基因序列.测序结果显示：该开放阅读框基因的序列长度为1 071 bp,编码356个氨基酸残基;该序列登录Genbank（GQ：851626）,序列比对结果显示,锯缘青蟹精氨酸激酶与凡纳滨对虾（...     

Functional link between ataxia-telangiectasia and Nijmegen breakage syndrome gene products

Ataxia-telangiectasia (A-T) and Nijmegen breakage syndrome (NBS) are recessive genetic disorders with susceptibility to cancer and similar cellular phenotypes. The protein product of the gene responsible for A-T, designated ATM, is a member of a family of kinases characterized by a carboxy-terminal phosphatidylinositol 3-kinase-like domain. The NBS1 protein is specifically mutated in patients with Nijmegen breakage syndrome and forms a complex with the DNA repair proteins Rad50 and Mrel1. Here we show that phosphorylation of NBS1, induced by ionizing radiation, requires catalytically active ATM. Complexes containing ATM and NBS1 exist in vivo in both untreated cells and cells treated with ionizing radiation. We have identified two residues of NBS1, Ser 278 and Ser 343 that are phosphorylated in vitro by ATM and whose modification in vivo is essential for the cellular response to DNA damage. This response includes S-phase checkpoint activation, formation of the NBS1/Mrel1/Rad50 nuclear foci and rescue of hypersensitivity to ionizing radiation. Together, these results demonstrate a biochemical link between cell-cycle checkpoints activated by DNA damage and DNA repair in two genetic diseases with overlapping phenotypes.     

Pb2 + 和Cd2 + 对日本三角涡虫DNA 损伤及损伤后修复的研究

摘要: 以日本三角涡虫( Dugesia japonica) 为实验材料,采用急性毒性实验研究Pb2 + 、Cd2 + 胁迫下日本三角涡虫体细 胞DNA 损伤情况以及绿豆浸出液对DNA 损伤的保护和修复机制。采用浓度为120 mg /L 的Pb( NO3 ) 2 和1 mg /L 的CdCl2 溶液分别处理涡虫,紫外分光光度法和琼脂糖凝胶电泳检测24 h 后三角涡虫DNA 损伤情况。同时增加 由绿豆浸出液进行修复的2 组对照以研究绿豆对于DNA 重金属损伤后的修复作用和效果。结果表明,Pb2 + 、Cd2 + 胁迫使日本三角涡虫DNA 交联程度增加,并引起DNA 链的断裂; 绿豆浸出液对于由Cd2 + 胁迫引起的DNA 损伤修 复作用较好。而对于Pb2 + 胁迫,在绿豆浸出液与Pb2 + 同时培养的对照组中,推测该浸出液可能使Pb2 + 形成沉淀从 而减小Pb2 + 浓度,因此使DNA 损伤修复具有较好效果,对已经由Pb2 + 胁迫造成损伤的DNA,修复作用不大。     

Separase-mediated cleavage of cohesin at interphase is required for DNA repair

Sister chromatids are held together by cohesins. At anaphase, separase is activated by degradation of its inhibitory partner, securin. Separase then cleaves cohesins, thus allowing sister chromatid separation. Fission yeast securin (Cut2) has destruction boxes and a separase (Cut1) interaction site in the amino and carboxyl terminus, respectively. Here we show that securin is essential for separase stability and also for proper repair of DNA damaged by ultraviolet, X-ray and gamma-ray irradiation. The cut2(EA2) mutant is defective in the repair of ultraviolet damage lesions, although the DNA damage checkpoint is activated normally. In double mutant analysis of ultraviolet sensitivity, checkpoint kinase chk1 (ref. 9) and excision repair rad13 (ref. 10) mutants were additive with cut2(EA2), whereas recombination repair rhp51 (ref. 11) and cohesin subunit rad21 (ref. 12) mutants were not. Cohesin was hyper-modified on ultraviolet irradiation in a Rad3 kinase-dependent way. Experiments using either mutant cohesin that cannot be cleaved by separase or a protease-dead separase provide evidence that this DNA repair function of securin-separase acts through the cleavage of cohesin. We propose that the securin-separase complex might aid DNA repair by removing local cohesin in interphase cells.