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

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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.