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.     

博来霉素对肝癌细胞DNA损伤以及H2AX相互作用蛋白的影响

组蛋白H2AX是DNA损伤修复反应中的关键蛋白,磷酸化的H2AX可以作为DNA损伤早期检测的金标准.实验通过检测磷酸化H2AX的改变来研究博来霉素对肝癌细胞和正常肝细胞DNA损伤作用的差异,推测肿瘤细胞抗药性可能是通过H2AX磷酸化来介导的.利用免疫共沉淀的方法,对博来霉素刺激下肝癌细胞中的H2AX复合物进行了免疫印迹...     

Bidirectional resection of DNA double-strand breaks by Mre11 and Exo1

Repair of DNA double-strand breaks (DSBs) by homologous recombination requires resection of 5'-termini to generate 3'-single-strand DNA tails. Key components of this reaction are exonuclease 1 and the bifunctional endo/exonuclease, Mre11 (refs 2-4). Mre11 endonuclease activity is critical when DSB termini are blocked by bound protein--such as by the DNA end-joining complex, topoisomerases or the meiotic transesterase Spo11 (refs 7-13)--but a specific function for the Mre11 3'-5' exonuclease activity has remained elusive. Here we use Saccharomyces cerevisiae to reveal a role for the Mre11 exonuclease during the resection of Spo11-linked 5'-DNA termini in vivo. We show that the residual resection observed in Exo1-mutant cells is dependent on Mre11, and that both exonuclease activities are required for efficient DSB repair. Previous work has indicated that resection traverses unidirectionally. Using a combination of physical assays for 5'-end processing, our results indicate an alternative mechanism involving bidirectional resection. First, Mre11 nicks the strand to be resected up to 300 nucleotides from the 5'-terminus of the DSB--much further away than previously assumed. Second, this nick enables resection in a bidirectional manner, using Exo1 in the 5'-3' direction away from the DSB, and Mre11 in the 3'-5' direction towards the DSB end. Mre11 exonuclease activity also confers resistance to DNA damage in cycling cells, suggesting that Mre11-catalysed resection may be a general feature of various DNA repair pathways.     

光化学衍生检测玉米中4种黄曲霉毒素

建立了HPLC-免疫亲和柱净化,柱后光化学衍生检测玉米中4种黄曲霉毒素B1,B2,G1,G2的分析方法。玉米样品提取、浓缩、过滤后经免疫亲和柱净化,液相色谱分离后采用光化学衍生器对黄曲霉毒素B1,B2,G1,G2进行在线衍生,通过配制荧光检测器的液相色谱同时检测玉米中B1,B2,G1,G2这4种黄曲霉毒素。结果表明,4种黄曲霉毒素B1,B2,G1,G2的色谱分析时间在6～11min内完成,检测定量限分别为:0.10,0.03,0.10,0.03μg/kg,完全符合并高于欧盟检测标准定量限,通过光化学衍生器在线衍生将B1、G1的分析定量限提高到2.7倍和3.6倍。玉米基质中B1,B2,G1,G2在添加水平为10,3,10,3μg/L时其回收率分别为:90.3%,85.6%,93.5%,92.8%。其线性范围分别为0～20μg/L,0～6μg/L,0～20μg/L,0～6μg/L,RSD 分别为 2.3%,1.5%,2.7%,3.2%。文章建立了分析玉米中4种黄曲霉毒素B1,B2,G1,G2的分析方法,经验证该方法定性准确,定量灵敏度高,可同时检测出玉米中4种黄曲霉毒素B1,B2,G1,G2含量。     

Defective DNA single-strand break repair in spinocerebellar ataxia with axonal neuropathy-1

Spinocerebellar ataxia with axonal neuropathy-1 (SCAN1) is a neurodegenerative disease that results from mutation of tyrosyl phosphodiesterase 1 (TDP1). In lower eukaryotes, Tdp1 removes topoisomerase 1 (top1) peptide from DNA termini during the repair of double-strand breaks created by collision of replication forks with top1 cleavage complexes in proliferating cells. Although TDP1 most probably fulfils a similar function in human cells, this role is unlikely to account for the clinical phenotype of SCAN1, which is associated with progressive degeneration of post-mitotic neurons. In addition, this role is redundant in lower eukaryotes, and Tdp1 mutations alone confer little phenotype. Moreover, defects in processing or preventing double-strand breaks during DNA replication are most probably associated with increased genetic instability and cancer, phenotypes not observed in SCAN1 (ref. 8). Here we show that in human cells TDP1 is required for repair of chromosomal single-strand breaks arising independently of DNA replication from abortive top1 activity or oxidative stress. We report that TDP1 is sequestered into multi-protein single-strand break repair (SSBR) complexes by direct interaction with DNA ligase IIIalpha and that these complexes are catalytically inactive in SCAN1 cells. These data identify a defect in SSBR in a neurodegenerative disease, and implicate this process in the maintenance of genetic integrity in post-mitotic neurons.     

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.     

具有两种服务的负顾客M/(G1/G2)/1休假排队系统

考虑具有两种不同服务的负顾客M/(G1/G2)/1休假排队模型,正顾客接受第一种服务后以概率θ(0≤θ≤1)进行第二种服务,或者以概率1-θ离开系统.服务规则是先到先服务(FCFS).在正顾客接受两种服务的过程中均可能有负顾客到达,负顾客不接受服务,只抵消正在接受服务的顾客(RCH).休假策略为空竭服务单重休假(E,SV),通过补充变量法求得了稳态队长的概率母函数的随机分解结果.     

Nbs1 is essential for DNA repair by homologous recombination in higher vertebrate cells

Double-strand breaks occur during DNA replication and are also induced by ionizing radiation. There are at least two pathways which can repair such breaks: non-homologous end joining and homologous recombination (HR). Although these pathways are essentially independent of one another, it is possible that the proteins Mre11, Rad50 and Xrs2 are involved in both pathways in Saccharomyces cerevisiae. In vertebrate cells, little is known about the exact function of the Mre11-Rad50-Nbs1 complex in the repair of double-strand breaks because Mre11- and Rad50-null mutations are lethal. Here we show that Nbs1 is essential for HR-mediated repair in higher vertebrate cells. The disruption of Nbs1 reduces gene conversion and sister chromatid exchanges, similar to other HR-deficient mutants. In fact, a site-specific double-strand break repair assay showed a notable reduction of HR events following generation of such breaks in Nbs1-disrupted cells. The rare recombinants observed in the Nbs1-disrupted cells were frequently found to have aberrant structures, which possibly arise from unusual crossover events, suggesting that the Nbs1 complex might be required to process recombination intermediates.     

Inclusive Minimal Surfacein G2，n1

刻划了从Ｒｉｅｍａｎｎ面到复Ｇｒａｓｓｍａｎｎ流形的具有拓扑度＞２ｎ（ｇ－１）或′阶有限的一切可兼极小浸入。     

Crucial role for DNA ligase III in mitochondria but not in Xrcc1-dependent repair

Mammalian cells have three ATP-dependent DNA ligases, which are required for DNA replication and repair. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme, or Escherichia coli LigA, an NAD(+)-dependent ligase. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.     

具有两种服务的负顾客Mζ/(G1/G2)/1排队系统

考虑单服务台提供两种服务的负顾客Mζ/(G1/G2)/1排队模型,每个正顾客接受第一种服务后以概率θ(0≤θ≤1)进行第二种服务,或者以概率1-θ离开系统.服务规则是先到先服务(FCFS).在正顾客接受两种服务的过程中均可能有负顾客到达,负顾客不接受服务,只抵消正在接受服务的正顾客(RCH).通过补充变量法求得了系统稳态队长的概率母函数.     

肝癌细胞株HepG2的G1期、G2/M期细胞蛋白质组表达差异的研究

通过TdR(胸腺嘧啶核苷)调控人肝癌细胞株HepG2的细胞周期,运用双向电泳-图像分析-质谱技术研究肝癌细胞株HepG2的G1期、G2/M期全细胞蛋白质组表达的差异,探索参与肝癌细胞株HepG2的G1期、G2/M期调控相关的蛋白.以TdR诱导肝癌细胞株HepG2,分别得到同步化的G1期、G2/M期细胞,流式细胞术检测.采用比较蛋白质组学的研究技术(双向电泳、质谱分析)筛选G1期、G2/M期差异表达的蛋白质.结果流式细胞术检测显示TdR诱导后,分别获得(63.62±2.82)%的G2/M期同步化细胞,(75.24±0.17)%的G1期同步化细胞,通过双向电泳-图像分析-质谱技术得到21个差异表达的蛋白.其中G2/M期表达,G1期未见表达有10种蛋白;存在三倍以上的差异点11个:2种在G2期表达上调,9种在G1期表达上调.说明TdR阻断法能够获得很好的同步化细胞.质谱鉴定得到的这些差异表达的蛋白质涉及到细胞周期调控、DNA结合、转录调控、mRNA剪接、肽链合成起始、折叠以及细胞代谢等方面.     

RadA： A protein involved in DNA damage repair processes of Deinococcus radiodurans R1

RadA is highly conserved in bacteria and belongs to the RecA/RadA/Rad51 protein su-perfamily found in bacteria,archaea and eukarya. In Archaea,it plays a critical role in homologous re-combination process due to its RecA-like function. In Escherichia coli,it takes part in conjugational recom-bination and DNA repair but is not as important as that of archaea. Using PSI-BLAST searches,we found that Deinococcus radiodurans RadA had a higher similarity to that of bacteria than archaea and eukarya. Disruption of radA gene in D. radiodurans resulted in a modestly decreased resistance to gamma radiation and ultraviolet,but had no effect on the resistance to hydrogen peroxide. Complementa-tion of the radA disruptant by both E. coli radA and D. radiodurans radA could fully restore its resistance to gamma radiation and ultraviolet irradiation. Further domain function analyses of D. radiodurans RadA showed that the absence of the zinc finger domain resulted in a slightly more sensitive phenotype to gamma and UV radiation than that of the radA mutant,while the absence of the Lon protease domain exhib-ited a slightly increased resistance to gamma and UV radiation. These data suggest that D. radiodurans RadA does play an important role in the DNA damage repair processes and its three different domains have different functions.     

C-terminal domain of Chk1 regulates its subcellular location and kinase activity for DNA repair

Effector kinase Chk1 is an evolutionarily conserved protein kinase. It is a key mediator linking the mechanisms that monitor DNA integrity to components of the cell cycle engine. In this study, recombinant vectors pEGFP-C1-Chk1/C 288/C 334/C 368 were constructed and transfected into HeLa cells to study the effect of the Chk1 regulatory domain on the regulation of subcellular Chk1 location in response to DNA damage. We found that DNA damage-induced nuclear accumulation is regulated by 34 amino acids (334–368) in the C-terminal regulatory domain. Recombinant vectors pXJ41-Chk1/C 288/C 334/C 368 were co-transfected with reporter plasmid pEGFP-N2 into HeLa cells to study the repair abilities of the different human Chk1 truncation mutants. In addition, recombinant vectors were transfected into HeLa cells to study the effects of the different truncation mutants on the cell cycle. Furthermore, to study the kinase activity of the different truncation mutants, Ser216 phosphorylation of Cdc25C was studied by Western blot analysis. We found that the enzymatic activity of C 368, missing the 108 C-terminal amino acids (368–476), was higher than that of full-length Chk1, and C 368 delayed the cell cycle progression. The enzymatic activity of C 334, missing the 142 C-terminal amino acids (334–476), was equivalent to that of full-length Chk1. C 288, missing the 188 C-terminal amino acids (288–476), had almost no enzymatic activity, suggesting that the regulatory domain contains both inhibitory and regulatory elements. This study provides useful information for further research on Chk1 function.