Functional and structural investigations of human replisomes assembled from purified proteins

DNA replication is a fundamental biological process for the transmission of genetic information from one generation of cells to the next in each cell division. Errors or abnormalities during replication can lead to mutations and genome instability, which is a hallmark of cancer. The complex and highly regulated molecular machinery responsible for an accurate and efficient replication is known as the replisome. At the heart of the eukaryotic replisome is the CMG (CDC45-MCM-GINS) helicase that unwinds the parental DNA duplex to generate single-stranded DNA for initiation of DNA synthesis by Pol α -primase and the replication of both strands by the replicative polymerases Pol ε and Pol δ. Replisomes contain multiple additional proteins including AND-1, CLASPIN and TIMELESS-TIPIN that function in replication and various replication-coupled processes. Biochemical reconstitution of DNA replication is a powerful approach to study the mechanism and structures of replisomes. Reconstitution of functional budding yeast replisomes has uncovered important aspects of the eukaryotic replication machinery including how the replisome achieves rapid DNA replication, how it responds DNA damage and how it navigates chromatin. Despite the significance of biochemical reconstitution and the critical importance of replication in human cells, an *in vitro* DNA replication system that utilises purified human proteins has not been established. Here, by developing the first *in vitro* human DNA replication system, I have investigated the requirements for efficient leading and lagging strand replication. Importantly, this system has enabled me to address the functions of several replisomes components in human DNA replication in isolation. In addition, reconstitution of functional human replisomes resulted in collaborative work that succeeded in determining the cryo-EM structure of a human replisome comprising CMG, TIMELESS-TIPIN, AND-1 and Pol ε bound to replication fork DNA. Here, we showed that how TIMELESS-TIPIN, AND-1 and Pol ε associate with CMG and provided structural insights into human replisome organisation. Finally, I have discussed the applications of the *in vitro* human DNA replication system.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Bariş, Yasemin

Advisor dc:contributor.advisor

• Yeeles, Joseph TP

• DNA replication, Replisome, DNA, CMG, biochemical reconstitution,

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/fa2278c9-9225-4fdf-b5e6-c69da5d9ce3e/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.99088

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/352939