Control of Programmed Replication fork Arrest by Chromosome Kissing

描述(申请人提供)：据报道，癌细胞中的主要DNA交易，如转录、发育、X染色体失活和染色体易位，都是由一种名为Reb1的MYB样终止子蛋白介导的染色体与染色体的接触控制的。我们最近在《细胞》杂志上发表的一篇论文中表明，分裂酵母中的染色体亲吻也通过控制序列特定的、生理编程的分叉停止来控制复制分叉的移动。这一发现提出了一些主要问题：(I)哪些蛋白质参与控制染色体亲吻，它们的作用机制是什么？(Ii)程序分叉停止的机制是什么？在回答第一个问题的背景下，我们发现分裂酵母的两个染色质重塑蛋白调节程序性分叉停顿。这意味着，这些重构体通过调节染色体亲吻和复制末端周围的核小体部署来实现这一点。这项提议的目标之一是在全基因组搜索中发现调节染色体接吻的蛋白质，并试图了解它们的作用机制。这项提议的另一个重要目标是通过测试通过单向抑制MCM2-7解旋酶而发生的假设来揭示程序性分叉抑制的作用机制。程序性分叉抑制控制其他染色体交易，如重组、基因沉默和转录通道，并处于复制和其他过程的中间阶段。最后，提出了定位全基因组依赖于Reb1的复制末端的实验。这一目标的目的是检验两个假说：(I)自然发生的非正则弱位点通过染色体亲吻和/或依赖DNA环的与强正则位点的相互作用而变得具有功能，这是这些长范围蛋白质-DNA相互作用的功能之一；(Ii)环依赖(或独立)分叉停止的功能是防止复制和转录之间的干扰。

DESCRIPTION (provided by applicant): Major DNA transactions such as transcription, development, X-chromosome inactivation and chromosomal translocations in cancer cells were reported to be controlled by chromosome to chromosome contacts called "chromosome kissing" mediated by a myb-like terminator protein called Reb1. We have recently shown in a paper to be published in Cell that chromosome kissing in fission yeast also controls replication fork movement by controlling sequence-specific, physiologically programmed fork arrest. This finding raises some major questions: (i) which proteins are involved in controlling chromosome kissing and what are their mechanisms of action?; (ii) what is the mechanism of programmed fork arrest? In the context of addressing the first question, we have discovered that two chromatin remodeling proteins of fission yeast modulate programmed fork arrest. The implication is that these remodelers do this by modulating chromosome kissing and nucleosome disposition about the replication termini. One of the goals of this proposal is to discover in a genome-wide search the proteins that modulate chromosome kissing and try to understand their mechanism of action. Another important goal of this proposal is to uncover the mechanism of action of programmed fork arrest by testing the hypothesis that this happens by unidirectional arrest of the MCM2-7 helicase. Programmed fork arrest controls other chromosome transactions such as recombination, gene silencing and transcriptional passage and is at the interphase of replication and other processes. Finally, experiments are proposed to localize genome-wide Reb1-dependent replication termini. The objectives of this goal is to test two hypotheses: (i) naturally occurring, noncanonical weak sites are rendered functional by chromosome kissing and/or DNA looping -dependent interaction with strong canonical sites and that is one function of these long range protein- DNA interactions; (ii) a function of looping-dependent (or independent) fork arrest is to prevent interference between replication and transcription.