Molecular mechanisms at the crossroads between DNA replication and chromosome cohesion establishment

DNA复制和染色体内聚力建立之间十字路口的分子机制

• 批准号: 417702356
• 负责人: Dr. Daniel Grabarczyk
• 金额: --
• 依托单位: Rudolf-Virchow-Zentrum - Center for Integrative and Translational Bioimaging
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2021-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/417702356?language=en
• 关键词: Molecular; mechanisms; crossroads; between; DNA

In eukaryotic organisms, DNA replication is a tightly regulated process which is connected to cellular signalling, transfers epigenetic information and is coordinated with the higher order structural organisation of the DNA but must also respond to damage and blocks on DNA. Any defects in these replication-associated processes will result in DNA replication stress and genomic instability, both starting points for the formation of cancer. Despite their fundamental physiological and pathological importance, it is still unclear how all these processes are orchestrated at the replication fork. Particularly unclear is how the two newly replicated chromatids are linked together at the replication fork in a process called cohesion, which is critical to separate each chromatid into a separate daughter cell at mitosis. To understand how cohesion is established, we will employ structural and functional studies on the Chl1, Tof1-Csm3 and Ctf18-RFC proteins, three replisome-associated factors thought to work together to establish chromosome cohesion at the replication fork. These proteins have been implicated in the formation and persistence of colorectal cancer and melanoma formation. Additionally, Chl1 deficiency is the cause of the severe developmental genetic disorder Warsaw breakage syndrome, and the protein is exploited by the human papillomavirus. Ctf18-RFC is an alternative processivity clamp loader with a unique module composed of the C-terminus of Ctf18 and the Dcc1-Ctf8 complex. This module binds to DNA and the leading strand DNA polymerase ε. To understand the function of these interactions with respect to linking replication to cohesion we will determine the architecture of Ctf18-RFC complexes with DNA and DNA polymerase ε. X-ray crystallographic snapshots of minimal complexes will be combined with site-directed mutagenesis and electron microscopy data to decipher the architecture of these large complexes. We will simultaneously pursue a full biochemical and biophysical functional analysis to elucidate how formation of each complex regulates the formation of the other complex and the enzymatic activity of each protein. Ctf18-RFC is thought to interact with Chl1, a member of the mechanistically elusive XPD-family of helicases with poorly defined physiological substrates. To understand the function of Chl1 in cohesion establishment we aim to validate and map the putative interaction between Chl1 and Ctf18-RFC. To gain insight into the cellular substrates of Chl1, we will generate a DNA substrate interaction profile for Chl1 and probe the molecular basis of this interaction by characterising mutations which disrupt binding to subsets of substrates. Our work will shed light on how both Ctf18-RFC and Chl1 establish sister chromatid cohesion at the replication fork, and provide insight into cohesion-related pathologies and future therapeutic approaches.

在真核生物中，DNA复制是一个严格调控的过程，与细胞信号传导有关，传递表观遗传信息，并与DNA的高级结构组织协调，但也必须对DNA上的损伤和阻断做出反应。这些复制相关过程中的任何缺陷都会导致DNA复制应激和基因组不稳定，这两者都是癌症形成的起点。尽管它们在生理学和病理学上都很重要，但目前还不清楚所有这些过程是如何在复制叉上协调的。特别不清楚的是，两个新复制的染色单体是如何在复制叉处连接在一起的，这一过程称为凝聚力，这对于在有丝分裂时将每个染色单体分离成一个单独的子细胞至关重要。为了了解凝聚力是如何建立的，我们将采用Chl 1，Tof 1-Csm 3和Ctf 18-RFC蛋白的结构和功能研究，这三个复制体相关因子被认为共同作用，在复制叉处建立染色体凝聚力。这些蛋白质与结直肠癌和黑色素瘤的形成和持续存在有关。此外，Chl 1缺乏是严重发育遗传性疾病华沙断裂综合征的原因，该蛋白质被人乳头瘤病毒利用。Ctf 18-RFC是一种替代的持续合成性夹钳加载器，具有由Ctf 18的C末端和Dcc 1-Ctf 8复合物组成的独特模块。该模块与DNA和前链DNA聚合酶ε结合。为了理解这些相互作用在连接复制和内聚方面的功能，我们将确定Ctf 18-RFC与DNA和DNA聚合酶ε的复合物的结构。最小复合物的X射线晶体学快照将与定点诱变和电子显微镜数据相结合，以破译这些大复合物的结构。我们将同时进行全面的生物化学和生物物理功能分析，以阐明每个复合物的形成如何调节其他复合物的形成和每个蛋白质的酶活性。Ctf 18-RFC被认为与Chl 1相互作用，Chl 1是具有不明确的生理底物的解旋酶的机械难以捉摸的XPD家族的成员。为了理解Chl 1在内聚建立中的功能，我们的目标是验证和映射Chl 1和Ctf 18-RFC之间的假定相互作用。为了深入了解Chl 1的细胞底物，我们将生成Chl 1的DNA底物相互作用谱，并通过表征破坏与底物子集结合的突变来探测这种相互作用的分子基础。我们的工作将阐明Ctf 18-RFC和Chl 1如何在复制叉处建立姐妹染色单体凝聚力，并提供对凝聚力相关病理学和未来治疗方法的见解。