Homologous recombination and gene amplification induced by DNA replication fork inhibition

在本项目开始之前，我们已经发现（1）在E.在大肠杆菌中，复制叉末端（Ter）的DNA复制叉阻断事件激活附近姐妹染色体区域的同源重组，将这些区域转化为重组热点Hot，和（2）在S.在酿酒酵母中，Fobl蛋白是在每个核糖体RNA基因（rDNA）重复序列中称为RFB的DNA复制叉屏障位点阻断DNA复制所必需的。通过观察fob 1-突变体中rDNA之间的重组和rDNA的扩增都没有发生，rDNA的扩增和rDNA的重组都需要在RFB位点上发生复制阻断事件，本项目的后续研究确定了以下几点：（1）在大肠杆菌的TerB位点附近的“热”区之一HotA;发现大肠杆菌染色体也可能通过滚环型复制扩增;扩增的程度，发生在10%的细胞群体中 ...更多信息 平均高达400倍左右。（2）虽然已经知道Sir 2是酵母中沉默蛋白之一，它是抑制PolII依赖性表达和rDNA重复内重组所必需的，但其机制仍然不确定。我们发现，sir 2缺陷的条件下，抑制特定的PolII依赖性双向转录从启动子E-pro，位于35 S rDNA和5S rDNA之间，被废除。这导致粘连蛋白从染色体DNA中解离，并降低姐妹染色体之间的结合，导致rDNA重复序列之间的不等重组激活，并改变rDNA的拷贝数。(3)In fobl突变体的rDNA拷贝数固定在较高水平。为了阐明这种维持机制，我们收集了6个突变体，其中rDNA拷贝数急剧下降，只有在fobl-条件下。我们发现，所有六个突变发生在凝聚素亚基基因。对凝聚素在rDNA区域的定位进行了序列分析，结果表明凝聚素与S期的RFB位点存在Fobl依赖性关联，这表明凝聚素是维持长rDNA串联阵列所必需的。Shinohara等人试图鉴定与减数分裂特异性酵母RecA同源物Dmcl相关的新蛋白因子，并发现了两个新蛋白，以前称为Mei 5和Sae 3蛋白。他们发现三种蛋白质形成一个复合物，并在减数分裂特异性重组中发挥作用，因为它们在减数分裂中与染色体的结合是相互依赖的。少

Before starting this project, we had already found that (1) in E. coli, DNA replication fork blocking event at a replication fork terminus (Ter) activates homologous recombination at the nearby sister chromosomal regions, converting these into recombinational hotspots, Hot, and (2) in S. cerevisiae, Fobl protein is required for blocking DNA replication at DNA replication fork barrier site called RFB in each ribosomal RNA gene (rDNA) repeat. From the observation that, in fob1-mutant, both processes, recombination between rDNA and amplification of rDNA, did not occur, replication blocking event at RFB site was required for rDNA amplification as well as rDNA recombination.From the subsequent research in this project, the following have been established ;(1)One of the "Hot" regions, HotA, near the TerB site of the E. coli chromosome was found to be also amplified probably through rolling circle type replication; the degree of the amplification, which occurred in 10 % of the cell population … More , was as high as approximately 400-fold on average.(2)Although it was already known that Sir2, one of the silencing proteins in yeast, is required for suppressing both PolII-dependent expression and recombination within rDNA repeats, the mechanism remained undetermined. We found that under sir2 defective conditions, suppression of specific PolII-dependent bi-directional transcriptions from the promoter E-pro, located between 35S rDNA and 5S rDNA, was abrogated. This results in the dissociation of cohesin from the chromosomal DNA and decreases the association between sister-chromosomes, resulting in the activation of unequal recombination between rDNA repeats and changing the copy number of rDNA.(3)In fobl mutant, the copy number of rDNA is fixed at a high level. In order to elucidate this maintenance mechanism, we collected six mutants in which the rDNA copy number was drastically decreased only under fobl- conditions. We found that all six mutations occurred in condensin subunit genes. Subsequence analysis of localization of condensin in rDNA region revealed that condensin was Fobl-dependently associated with RFB site in S phase, thereby suggesting that condensin is essential for maintenance of a long rDNA tandem array.Shinohara's group tried to identify new protein factors associated with Dmcl, a meiosis-specific yeast, RecA homologue, and found two new proteins, previously called Mei5 and Sae3 proteins. They showed three proteins form a complex and play a role in meiotic specific recombination, because their binding to chromosome in meiosis is mutually dependent. Less