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

在真核生物中，DNA复制是一个严格调控的过程，与细胞信号有关，传递表观遗传信息，与DNA的高阶结构组织协调，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将导致DNA复制压力和基因组不稳定，这两者都是癌症形成的起点。尽管它们在生理和病理上具有基本的重要性，但目前仍不清楚所有这些过程是如何在复制分叉上进行协调的。尤其不清楚的是，这两个新复制的染色单体是如何在复制分叉上连接在一起的，这一过程被称为内聚力，这是在有丝分裂时将每个染色单体分离成单独的子细胞的关键。为了了解凝聚力是如何建立的，我们将对CHL1、Tof1-Csm3和Ctf18-RFC蛋白进行结构和功能研究，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。这些蛋白与结直肠癌和黑色素瘤的形成和持续存在有关。此外，CHL1缺乏是严重的发育遗传性疾病华沙破裂综合征的原因，该蛋白被人类乳头瘤病毒利用。Ctf18-RFC是一种替代加工性夹具加载器，具有独特的模块，由Ctf18的C端和Dcc1-Ctf8复合体组成。该模块与脱氧核糖核酸和领先的脱氧核糖核酸聚合酶ε结合。为了了解这些相互作用在将复制与凝聚力联系起来方面的功能，我们将确定CTF18-RFC与脱氧核糖核酸和脱氧核糖核酸聚合酶ε的复合体的结构。微型复合体的X射线晶体快照将与定点突变和电子显微镜数据相结合，以破译这些大型复合体的结构。我们将同时进行全面的生化和生物物理功能分析，以阐明每个复合体的形成如何调节另一个复合体的形成和每个蛋白质的酶活性。Ctf18-RFC被认为与CHL1相互作用，CHL1是机械上难以捉摸的XPD解旋酶家族的成员，其生理底物定义不明确。为了了解CHL1在凝聚力建立中的功能，我们的目标是验证和定位CHL1与Ctf18-RFC之间的可能相互作用。为了深入了解CHL1的细胞底物，我们将生成CHL1的DNA底物相互作用谱，并通过表征破坏与底物亚群结合的突变来探索这种相互作用的分子基础。我们的工作将阐明Ctf18-RFC和CHL1如何在复制分叉建立姐妹染色单体凝聚力，并为凝聚力相关的病理和未来的治疗方法提供洞察力。

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.