Role of polyhomeotic and the cohesin protein Pds5 in silencing and S phase

为了正常发育，基因必须在它们应该关闭的地方保持关闭(沉默)。沉默在发育中起着至关重要的作用，因为它可以防止基因过早开启或保持开启。这两种形式的不当调控都可能导致出生缺陷或癌症。由大约20种蛋白质组成的聚梳组蛋白质共同作用，使启动基因的蛋白质无法接触到目标基因的DNA。我们正在研究其中的一种蛋白质，称为多态同源异体。在之前由NSERC资助的工作中，一名研究生发现多同源异体与一种蛋白质(Pds5)有关，该蛋白质的已知作用是在细胞分裂前将新复制的DNA保持在一起。另一名学生使用基因分析表明，Pds5在沉默方面具有意想不到的作用。这项提议的目的之一是确定Pds5在沉默中的分子作用。在另一项单独的研究中，我的学生使用遗传分析表明，正常的细胞分裂需要多核分裂，并与Pds5一起工作。非常令人惊讶的是，复制DNA的细胞分裂阶段在多染色体突变体中加速。这项提议的另一个目的是理解为什么会发生这种情况。最可能的原因是多核生物中的突变使蛋白质机制更容易复制DNA，或者突变使复制机制可以获得更多的DNA复制起点。PDS5是由制造DNA的蛋白质机器招募的。我们想要测试的假设是，Pds5从新复制的DNA中招募多个同源基因。要做到这一点，我们必须开发一种系统，以不破坏蛋白质与DNA的相互作用的方式来纯化这种新合成的DNA。这是该提案的第三个目标。这项研究将确定PDS5新角色(S)的分子基础，并有可能为沉默的分子机制提供重要的见解。

For normal development to occur, genes must be kept off (silent) where they are supposed to be off. Silencing plays a vital role in development because it prevents genes from being turned on too early, or from being kept on. Both forms of misregulation can lead to birth defects, or to cancer. A group of about 20 proteins, called the Polycomb group proteins work together to make a the DNA of a target gene inaccessible to the proteins that turn genes on. We are studying one of these proteins, called Polyhomeotic. In previously work previously funded by NSERC, a graduate student discovered that Polyhomeotic associates with a protein (Pds5) whose known role is in holding newly replicated DNA together before cell division. Another student has shown using genetic analysis that Pds5 has an unexpected role in silencing. One of the aims of this proposal is to determine the molecular role for Pds5 in silencing. In a separate study, my students have shown using genetic analysis that Polyhomeotic is required for normal cell division, and works together with Pds5. Very surprisingly, the stage of cell division where DNA is replicated is speeded up in Polyhomeotic mutants. Another aim of this proposal is to understand why this occurs. The most likely reasons are that mutations in Polyhomeotic make it easier for the protein machinery to replicate DNA, or that mutations make more starting points for DNA replication available to the replication machinery. Pds5 is recruited by the protein machinery that makes DNA. We want to test the hypothesis that Pds5 recruits Polyhomeotic to newly replicated DNA. To do this, we have to develop a system for purifying this newly synthesized DNA in a way that does not disrupt the interaction of proteins with DNA. This is the third aim of the proposal. This research will identify the molecular basis for new role(s) for Pds5, and has the potential to provide important insight about the molecular mechanisms of silencing.