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) 可能由电离辐射、烷基化损伤和复制引起，包括滞后链中间体的不当处理。 DNA 断裂可能是染色体不稳定以及程序性遗传修饰的重要根源。细胞拥有复杂的系统来处理 DSB，包括 DNA 修复和检查点抑制，以增加修复的机会。染色体中的 DSB 会导致酵母 G2/M 边界处的检查点停滞，从而提供进一步的修复机会。 DSB 通过同源重组、末端连接以及断裂以外同源区域的单链退火进行修复。几乎所有生物体都表现出这些修复过程以及检查点停滞。这些过程中的缺陷通常与包括癌症在内的人类疾病有关。 DNA 末端必须经过处理，以实现重组和单链退火的同源相互作用。端接仅涉及局部核酸酶降解，从而能够在仅少数碱基的微同源性处发生相互作用，端接需要蛋白质的 Ku 和 RAD50/MRE11/XRS2 (R/M/X) 复合物。此外，R/M/X 复合物在核酸酶末端加工中发挥作用，提供重组底物。 Ku 复合体在断裂末端结合，可防止断裂末端的过度加工并提供末端连接。我们正在研究 DSB 在各种突变体中的后果以及处理 DSB 的机制。我们的方法已扩展到考虑染色体和染色质组织背景下的修复。虽然 DNA 是染色体的核心组成部分，但有关 DNA 断裂修复、修复系统和染色质组织之间关系的信息却很少。我们还继续研究影响各种双链断裂剂敏感性的基因特征。 染色质具有修复和重组功能。 S 期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的细胞对甲磺酸甲酯和喜树碱等DNA损伤剂高度敏感，表明双链断裂(DSB)修复可能存在缺陷。为定量确定的、粘性末端断裂结构的修复而开发的测定表明，在RCAF/CAF-1双突变体中，DSB诱导的质粒：染色体重组减少了大约10倍。体内染色体和质粒靶标的重组缺陷相似，表明不涉及抑制性染色质结构。与这些观察结果一致，电离辐射引起的杂合性丢失（LOH）在突变体中被消除。 NHEJ 修复能力和准确性介于野生型水平和 NHEJ 缺陷型 yku70 和 rad50 突变体水平之间。 NHEJ 中的缺陷（而非同源重组）可以通过删除 HMR-a1（a1/α2 转录抑制复合物的一个组成部分）来挽救。这些发现与当染色质组装减少时沉默交配位点部分去抑制的观察结果一致。这些结果表明 CAF-1 和 RAF 在重组中具有复制后修复功能，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。 鉴定抗双链断裂剂所需的基因。 细胞对 DNA 损伤剂的反应涉及许多基因和途径。我们采取了系统的方法来识别影响电离辐射生存/生长反应的所有基因。为了识别 DSB 诱导后维持遗传完整性所需的新重组或检查点基因，我们之前检查了 3670 个非必需基因，以了解单次急性剂量 IR 后二倍体纯合突变对生长和/或致死率的影响。我们最初发现了 107 个抗辐射所需的新基因。其中许多似乎影响复制、重组和检查点功能，并且超过 50% 与人类基因具有同源性，其中 17 个与癌症有关。我们现在已经完成了这项研究，总共找到了 169 个耐辐射所需的新基因。因此，大约 4% 的非必需基因是耐受 IR 损伤所必需的。随着这一筛选的完成，我们首次确定了耐受红外线损伤所需的非必需基因的总补充。大约 90% 的基因影响对其他 DNA 损伤剂的抵抗力，包括博来霉素、阿霉素、甲磺酸甲酯 (MMS)、羟基脲 (HU)、喜树碱和紫外线 (UV)。 利用现有的遗传和蛋白质组数据库，发现其中许多基因在损伤反应网络中与转录因子 Ccr4 相互作用，转录因子 Ccr4 是 CCR4NOT 和 PAF-CDC73 转录复合物的核心成分。这两个复合体的单个成员的缺失使得细胞作为二倍体而非单倍体对IR的致死作用敏感，这表明二倍体G1细胞群是放射敏感的。与 G1 中的作用一致，与作为同步 G2 群体暴露的细胞相比，在 G1 中辐照的二倍体 ccr4 细胞显示出增强的致死率。此外，在 ccr4 细胞 IR 后发生了长时间的 RAD9 依赖性 G1 停滞，并且 CCR4 是 RAD9 上位组的成员，从而证实了 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，过渡 S 期的 ccr4 细胞会经历长时间的细胞周期停滞在 G2，随后发生细胞裂解。这种 S 期复制缺陷与 rad52 突变体所见的缺陷不同，因为与单独的 rad52 或 ccr4 突变体相比，rad52 ccr4 细胞显示出对 HU 的敏感性增加。这些结果表明，在辐射或复制应激后，细胞周期通过 G1 和 S 期的转变是 CCR4 依赖性的。