Replication Associated Genetic Rearrangements

描述（由申请人提供）：近年来积累的证据表明，复制分叉经常停止并需要修复才能重新启动。因此，重组过程为复制提供了基础，使整个染色体得以复制。我们对大肠杆菌dna复制解旋酶缺陷诱导的基因重排的研究揭示了对RadA/Sms蛋白的需求。在一些重组和DNA修复实验中，radA基因与编码Holliday连接加工蛋白的基因ruvABC和recG起着冗余的作用。因此，RadA很可能起到稳定或处理重组中间体的作用。RadA是一种普遍存在的真菌性蛋白，其序列与RecA/Rad51Dmc 1链交换因子和Lon蛋白酶相似。我们的第一个具体目标是表征纯化的RadA蛋白的生化特性，包括DNA结合、链交换、蛋白酶和重组中间裂解活性。对其生化特性的阐明有助于揭示RadA在重组和DNA修复中的作用。细菌中复制叉修复的三个方面仍未完全了解：复制检查点反应、染色体和蛋白质的细胞定位对修复的影响以及修复过程中组装和拆卸反应的协调。我们的第二个目标是通过对大肠杆菌中复制叉修复的遗传分析来澄清这些领域。对羟基脲的敏感性，核糖核苷酸还原酶抑制剂已被用于鉴定酵母叉修复和复制检查点突变体。我们对大肠杆菌hu敏感突变体的中试筛选发现了一种可能调节细胞分裂检查点的信号转导蛋白，并提出进一步表征该突变体以验证这一假设。一些具有reca合成DNA损伤或活力表型的有趣突变体已经被分离出来并将被表征。提出了传统的和基于基因组的突变分析。已知控制复制起始和染色体定位的蛋白质也将被研究对叉修复的影响。我们的第三个目标是进行微阵列分析大肠杆菌基因组转录对复制叉阻滞或损伤的反应。这些可能揭示了细胞对复制叉阻滞的反应中涉及的新功能。这一目的通过确定可能控制叉损伤耐受性或修复的候选基因来补充第二个目的。此外，在第二个目标中鉴定的突变体可以在复制停止条件下筛选对全局基因表达的影响。

DESCRIPTION (provided by the applicant): In recent years evidence has accumulated that replication forks frequently arrest and require repair to be restarted. Recombinational processes therefore underpin replication to permit replication of the entire chromosome. Our study of genetic rearrangements that are induced by defects in the DnaB replicative helicase of E. coli has revealed a requirement for the RadA/Sms protein. In several recombination and DNA repair assays, the radA gene plays a role redundant with ruvABC and recG, genes that encode Holliday junction processing proteins. RadA therefore most likely acts to stabilize or process recombination intermediates. RadA is a ubiquitous eubacterial protein that shares sequence similarity to the RecA/Rad51Dmc 1 strand exchange factors and to Lon protease. Our first specific aim is to characterize the biochemical properties of purified RadA protein tbr DNA binding, strand exchange, protease and recombination intermediate cleavage activities. The elucidation of its biochemical properties should reveal how RadA acts in recombination and DNA repair. Three areas of replication fork repair in bacteria remain incompletely understood: the replication checkpoint response, the influence of cellular localization of the chromosome and proteins on repair and the orchestration of assembly and disassembly reactions during repair. Our second aim seeks to clarify these areas by genetic analysis of replication fork repair in E. coli. Sensitivity to hydroxyurea, an inhibitor of Ribonucleotide reductase has been used to identify fork repair and replication checkpoint mutants in yeast. Our pilot screen for HU-sensitive mutants of E. coli has revealed a signal transduction protein that may regulate a cell division checkpoint and further characterization of this mutant is proposed to test this hypothesis. Several interesting mutants with recA-synthetic DNA damage or viability phenotypes have been isolated and will be characterized. Both traditional and genomic-based mutant analyses are proposed. Proteins known to control replication initiation and chromosome localization will also be investigated for effects on fork repair. Our third aim is to perform micro array analysis of E. coli genomic transcription in response to replication fork arrest or damage. These may reveal novel functions involved in the cellular response to replication fork arrest. This aim complements the second by identifying candidate genes that may control tolerance or repair of fork damage. Furthermore, mutants identified in the second aim can be screened for effects on global gene expression after conditions of replication arrest.