DNA helicase and replication factor functions in genome maintenance

摘要 基本DNA-DNA相互作用的改变可导致严重的多谱发育异常。 疾病、细胞非整倍性、基因组不稳定性和癌症。例如，DNA-DNA的不稳定 顺式（环状DNA分子的底部）的相互作用被认为会破坏染色质 DNA调控元件（增强子、启动子、绝缘子）的压缩和登记， 部署发展转录程序。或者，DNA-DNA相互作用的不稳定 反式（由染色体复制产生的姐妹染色单体之间）同时消除 姐妹篇的身份需要高保真染色体分离，也可以获得模板 无错误DNA修复所需的DNA。 粘着蛋白复合物对于顺式和反式DNA-DNA缔合都是至关重要的。因此， 途径突变直接导致发育程序的失调，导致严重的， 多谱出生缺陷疾病，如罗伯茨综合征（RBS）、科尔内利亚德兰格综合征 （CdLS）和华沙断裂综合征（WBS），也与许多形式的 癌症、非整倍体和遗传毒性敏感性。对粘着蛋白调节的复杂分析是， 绝大多数的粘附素沉积在DNA上，不依赖于核苷酸序列， 黏连蛋白可以在细胞周期的任何部分将任何两个DNA片段连接在一起。我们所知道的 Scc 2和Scc 4异源复合物对于所有粘附素沉积到DNA上是必需的， 序列、系链构象或细胞周期阶段。不幸的是，关于 Scc 2，4对DNA的募集作用甚至超过了粘着蛋白-分析同样受到以下发现的阻碍，即任何 DNA序列可以支持Scc 2，4募集和细胞周期的任何阶段。 这个修订后的R15更新提案是基于我的两个令人兴奋的发现， 实验室首先，我们最近确定Chl 1 DNA解旋酶作为Scc 2沉积的关键调节因子。在特定 目的1，我们将详细描述Scc 2沉积的基于染色质的上游调控， 我们预测将导致一个统一的模型，通过该模型，Scc 2在DNA中的募集发生在 转录、DNA复制和修复。在通过以前的R15奖励提供支持期间，我们还 成功地将顺式系链中的粘附素功能与反式系链中的粘附素功能分离（反之亦然）。 此外，我们发现，每一个都是由空间和时间上不同的机制调节的-- 其中包括PCNA。在修订后的R15更新提案的具体目标2中，我们将剖析 PCNA独立调节跨粘蛋白系链的分子基础。

ABSTRACT Alterations in basic DNA-DNA interactions can result in severe multi-spectrum developmental disorders, cell aneuploidy, genome instabilities and cancer. For instance, destabilization of DNA-DNA interactions in cis (at the base of a looped DNA molecule) are thought to abolish chromatin compaction and the registration of DNA regulatory elements (enhancers, promoters, insulators) that deploy developmental transcription programs. Alternatively, destabilization of DNA-DNA interactions in trans (between sister chromatids that arise by chromosome replication) simultaneously abolishes the identity of sisters required for high fidelity chromosome segregation and also access to template DNA required for error-free DNA repair. Cohesin complexes are critical for both cis and trans DNA-DNA associations. Thus, cohesin pathway mutations directly lead to deregulation of developmental programs that result in severe and multi-spectrum birth defect maladies such as Roberts Syndrome (RBS), Cornelia de Lange Syndrome (CdLS) and Warsaw Breakage Syndrome (WBS) and also correlate tightly with numerous forms of cancer, aneuploidy and genotoxic sensitivities. Complicating analyses of cohesin regulation is that the vast majority of cohesins are deposited onto DNA, independent of nucleotide sequence, such that cohesins can tether together any two DNA segments and in any part of the cell cycle. What we know is that the Scc2 and Scc4 heterocomplex is essential for all cohesin deposition onto DNA regardless of sequence, tether conformation or cell cycle stage. Unfortunately, less is known regarding the recruitment of Scc2,4 to DNA than even cohesin – analyses similarly impeded by findings that any DNA sequence can support Scc2,4 recruitment and in any stage of the cell cycle. This revised R15 Renewal Proposal is predicated on two exciting findings emanating from my lab. First, we recently identified Chl1 DNA helicase as a key regulator of Scc2 deposition. In Specific Aim 1, we will characterize in detail the chromatin-based upstream regulation of Scc2 deposition that we predict will result in unified model through which Scc2 recruitment to DNA occurs during transcription, DNA replication and repair. During support through a previous R15 award, we also succeeded in isolating cohesin functions in cis tethering from that of trans tethering (and vice versa). Moreover, we discovered that each is regulated by spatially and temporally distinct mechanisms - one of which involves PCNA. In Specific Aim 2 of this revised R15 Renewal Proposal, we will dissect the molecular basis through which PCNA independently regulates trans cohesin tethers.