Double-strand Breaks And Untargeted Dna Metabolic Events

双链断裂和非靶向 DNA 代谢事件

• 批准号: 7161811
• 负责人: MICHAEL A RESNICK
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ENVIRONMENTAL HEALTH SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7161811
• 关键词: DNA damage; DNA directed RNA polymerase; DNA repair; Saccharomyces cerevisiae; biotechnology; cell cycle; chemokine receptor; chromatin; fungal genetics; gene mutation; genetic recombination; genetic screening; histones; molecular biology information system; platelet activating factor; proteomics; radiation genetics; radiation resistance; radiation sensitivity; transcription factor

DNA double-strand breaks (DSB) can arise by ionizing radiation, alkylation damage, and replication, including improper processing of lagging strand intermediates. DNA breaks can be a powerful source of chromosome instability as well as programmed genetic modification. Cells have elaborate systems for dealing with DSBs, including DNA repair and checkpoint arrest to increase the opportunity for repair. DSBs in chromosomes lead to a checkpoint arrest at the G2/M boundary in yeast, which provides further opportunities for repair. DSBs are repaired through homologous recombination, end-joining , and by single-strand annealing at homologous regions beyond the breaks. Nearly all organisms exhibit these repair processes as well as checkpoint arrests. Defects in these processes are often associated with disease in humans including cancer. DNA ends must be processed to allow homologous interactions for recombination and single strand annealing. Endjoining involves only local nuclease degradation that enables interaction at microhomologies of only a few bases, The Ku and RAD50/MRE11/XRS2 (R/M/X) complexes of proteins are required for endjoining. In addition, the R/M/X complex functions in the nuclease processing of ends to provide recombination substrates. The Ku complex, which associates at the ends of breaks prevents excessive processing of broken ends as well as providing end joining. We are examining the consequences of DSBs in various mutants and the mechanisms of handling DSBs. Our approaches have been extended to consider repair in the context of chromosomes and chromatin organization. While DNA is the central component of chromosomes, there is little information about the relationship between DNA break repair, repair systems and chromatin organization. We have also continued studies on the characterization of genes that affect the sensitivity to a variety of double-strand breaking agents. Chromatin functions in repair and recombination. Assembly of new chromatin during S phase requires the histone chaperone complexes CAF-1 (Cac2p, Msi1p and Rlf2p) and RCAF (Asf1p plus acetylated histones H3 and H4). Cells lacking CAF-1 and RCAF are hypersensitive to DNA damaging agents such as methyl methanesulfonate and camptothecin, suggesting a possible defect in double-strand break (DSB) repair. Assays developed to quantitate repair of defined, cohesive-ended break structures revealed that DSB-induced plasmid:chromosome recombination was reduced approximately 10-fold in RCAF/CAF-1 double mutants. Recombination defects were similar with both chromosomal and plasmid targets in vivo, suggesting that inhibitory chromatin structures were not involved. Consistent with these observations, ionizing radiation-induced loss of heterozygosity (LOH) was abolished in the mutants. NHEJ repair proficiency and accuracy were intermediate between wildtype levels and those of NHEJ-deficient yku70 and rad50 mutants. The defects in NHEJ, but not homologous recombination, could be rescued by deletion of HMR-a1, a component of the a1/alpha2 transcriptional repressor complex. The findings are consistent with the observation that silent mating loci are partially derepressed when chromatin assembly is reduced. These results suggest a post-replicative repair function for CAF-1 and RCAF in recombination, possibly involving deposition of new histone octamers after DNA synthesis associated with strand exchange. Identification of genes required for resistance to double-strand breaking agents. The cellular response to DNA damaging agents involves many genes and pathways. We have taken a systematic approach to identifying all genes that impact on survival/growth response to ionizing radiation. In order to identify new recombination or checkpoint genes that are required for the maintenance of genetic integrity following induction of DSBs, we previously examined 3670 nonessential genes for the consequences of diploid homozygous mutations on growth and/or lethality following a single acute dose of IR. We initially found 107 new genes that were required for radiation resistance. Many of these appear to affect replication, recombination and checkpoint functions and >50% share homology with human genes including 17 implicated in cancer. We have now completed the study and a total of 169 new genes that are required for radiation toleration. Thus ~4% of the nonessential genes are required for toleration of IR damage. With the completion of this screen, we have determined for the first time the total complement of nonessential genes required for the toleration of IR damage. Approximately 90% of the genes affect resistance to other DNA damaging agents including bleomycin, doxorubicin, methyl methanesulfonate (MMS), hydroxyurea (HU), camptothecin and ultraviolet light (UV). Using existing genetic and proteomic databases, many of these genes were found to interact in a damage response network with the transcription factor Ccr4, a core component of the CCR4NOT and PAF-CDC73 transcription complexes. Deletions of individual members of these two complexes render cells sensitive to the lethal effects of IR as diploids, but not as haploids, indicating that the diploid G1 cell population is radiosensitive. Consistent with a role in G1, diploid ccr4 cells irradiated in G1 show enhanced lethality when compared to cells exposed as a synchronous G2 population. In addition, a prolonged RAD9-dependent G1 arrest occurred following IR of ccr4 cells and CCR4 is a member of the RAD9 epistasis group thus confirming a role for CCR4 in checkpoint control. Moreover, ccr4 cells that transit S phase in the presence of the replication inhibitor hydroxyurea (HU) undergo prolonged cell cycle arrest at G2 followed by cellular lysis. This S phase replication defect is separate from that seen for rad52 mutants since rad52 ccr4 cells show increased sensitivity to HU when compared to rad52 or ccr4 mutants alone. These results indicate that cell cycle transition through G1 and S phases is CCR4-dependent following radiation or replication stress.

DNA双链断裂（DSB）可以通过电离辐射，烷基化损伤和复制（包括滞后链中间体的处理不当）而产生。 DNA断裂可以是染色体不稳定性和编程遗传修饰的强大来源。细胞具有精心处理DSB的精心设计系统，包括DNA修复和检查点逮捕，以增加维修的机会。染色体中的DSB导致酵母中G2/m边界的检查站停滞，这提供了进一步的维修机会。 DSB通过同源重组，最终结合以及通过突破超出同源区域的单链退火来修复。几乎所有生物都表现出这些维修过程以及检查站逮捕。这些过程中的缺陷通常与包括癌症在内的人类中的疾病有关。 必须处理DNA末端以允许同源相互作用进行重组和单链退火。最终加入仅涉及局部核酸酶降解，该核酸酶降解只能在仅几个碱基的微观理学上进行相互作用，KU和RAD50/MRE11/XRS2（R/M/M/X）蛋白质的复合物是最终加入所必需的。此外，R/M/X复合物在末端的核酸酶处理中的功能提供重组底物。在断裂末端关联的KU综合体可阻止过度处理破裂的末端，并提供结束连接。我们正在研究各种突变体中DSB的后果以及处理DSB的机制。我们的方法已扩展到在染色体和染色质组织的背景下考虑维修。虽然DNA是染色体的核心组成部分，但关于DNA断裂修复，修复系统和染色质组织之间关系的信息很少。我们还继续研究影响对各种双链破裂剂的敏感性的基因的表征。 染色质在修复和重组中起作用。 新染色质的组装需要组蛋白伴侣复合物CAF-1（CAC2P，MSI1P和RLF2P）和RCAF（ASF1P加乙酰化组蛋白H3和H4）。缺乏CAF-1和RCAF的细胞对DNA损伤剂（例如甲基磺酸甲酯和Camptothecin）高度敏感，这表明双链断裂（DSB）修复中可能存在缺陷。开发的分析是为了量化定义的粘性断裂结构的修复，表明DSB诱导的质粒：在RCAF/CAF-1双突变体中，染色体重组降低了约10倍。重组缺陷与体内的染色体和质粒靶标都是相似的，这表明抑制性染色质结构不涉及。与这些观察结果一致，在突变体中废除了电离辐射诱导的杂合性损失（LOH）。 NHEJ修复能力和准确性介于野生型水平和NHEJ缺陷YKU70和RAD50突变体的水平之间。 NHEJ中的缺陷（但不是同源重组）可以通过缺失HMR-A1（A1/Alpha2转录抑制剂复合物的一个组成部分）来挽救。这些发现与观察到的观察结果一致：当染色质组装减少时，沉默的交配基因座被部分解压缩。这些结果表明在重组中CAF-1和RCAF的复制后修复功能，可能涉及与链交换相关的DNA合成后新组蛋白八聚体的沉积。 鉴定对双链破裂剂的抗性所需的基因。 细胞对DNA损伤剂的反应涉及许多基因和途径。我们采用了一种系统的方法来识别影响对电离辐射的生存/生长反应的所有基因。为了鉴定诱导DSB后维持遗传完整性所需的新的重组或检查点基因，我们先前检查了3670个非必需基因，以实现二倍体纯合突变对生长和/或单次急性剂量IR剂量后的生长和/或致命性的后果。我们最初发现了107个辐射抗性所需的新基因。其中许多似乎影响了复制，重组和检查点功能，> 50％与人类基因共享同源性，其中17个与癌症有关。现在，我们已经完成了研究，总共有169个新基因辐射耐受性。因此，〜4％的非必需基因是耐受IR损伤所必需的。随着该屏幕的完成，我们首次确定了IR损伤所需的非必需基因的总补体。大约90％的基因会影响对其他DNA损伤药物的耐药性，包括博来霉素，阿霉素，甲磺酸甲酯（MMS），羟基酸甲酯（HU），Camptothecin和Ultraviolet Light（UV）。 使用现有的遗传和蛋白质组学数据库，发现其中许多基因与转录因子CCR4相互作用，这是CCR4NOT和PAF-CDC73转录复合物的核心成分。这两种复合物中各个成员的删除使细胞对IR作为二倍体的致命作用敏感，但不是单倍体，表明二倍体G1细胞群是放射敏感的。与在G1中辐照的二倍体CCR4细胞在G1中的作用一致，与暴露为同步G2群体相比，G1中的二倍体CCR4细胞具有增强的致死性。此外，在CCR4细胞的IR和CCR4的IR之后，发生了延长的RAD9依赖性G1停滞，因此是Rad9 epitipasis组的成员，因此证实了CCR4在检查点控制中的作用。此外，在复制抑制剂羟基脲（HU）存在下转移S相的CCR4细胞在G2时经历了延长的细胞周期停滞，然后进行细胞裂解。该相位复制缺陷与Rad52突变体看到的缺陷是分开的，因为与单独的RAD52或CCR4突变体相比，RAD52 CCR4细胞对HU的敏感性增加。这些结果表明，在辐射或复制应力之后，通过G1和S相的细胞周期过渡是CCR4依赖性的。