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Functional and structural investigations of human replisomes assembled from purified proteins


Type

Thesis

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Authors

Bariş, Yasemin 

Abstract

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.

Description

Date

2023-04-20

Advisors

Yeeles, Joseph TP

Keywords

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

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge
Sponsorship
LMB Cambridge Trust International Scholarship