Establishing a method to monitor replicative polymerase usage genome-wide in human cells

Buist, David John (2019) Establishing a method to monitor replicative polymerase usage genome-wide in human cells. Doctoral thesis (PhD), University of Sussex.

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Abstract

The majority of DNA replication in eukaryotic cells is carried out by two replicative DNA polymerases: polymerases δ and ε (Pols δ and ε). Whilst both polymerases exhibit high specificity for deoxyribonucleotides, both frequently misincorporate ribonucleotides (rNTPs) which are rapidly removed via ribonucleotide excision repair (RER) by the heterotrimeric enzyme RNase H2.

In yeast, using mutant polymerases that incorporate elevated levels of rNTPs (and in the absence of the catalytic subunit of RNase H2 (RNase H2A)) unrepaired rNTPs serve as a molecular footprint of the replicative polymerases. This allows the determination of genome‐wide polymerase usage using a deep sequencing methodology that we have termed polymerase usage sequencing (Pu‐seq).

The aim of this project is to develop cell lines that can be used for Pu-­‐seq in human cells. Ultimately, this system could then be used to monitor DNA replication dynamics in human cells to aid in the study of replication stress responses. As in yeast, the development of the Pu‐seq system in human cells requires an absence of RNase H2 activity and mutant alleles of Pol δ and Polε that result in excess ribonucleotide incorporation.

This thesis describes the steps taken towards developing Pu‐seq in human cell lines; firstly, by establishing that in human cells expressing normal levels of p53, RNase H2A is an essential gene, then by developing mechanisms that allow for the shutoff of RNase H2 activity through the targeted downregulation of one of its accessory subunits, RNase H2C. Attempts to develop a novel method of quantifying genomic ribonucleotides are described in addition to showing that previously published protocols for assessing overall RNase H2 activity and genomic ribonucleotide presence provide the sensitivity required to screen candidate human Pu‐seq cell lines. Finally, progress made in screening mutations in Pols δ and ε that may cause increased ribonucleotide incorporation is described.

Item Type: Thesis (Doctoral)
Schools and Departments: School of Life Sciences > Sussex Centre for Genome Damage and Stability
Subjects: Q Science > QH Natural history > QH0301 Biology > QH0426 Genetics > QH0447 Genes. Alleles. Genome
Depositing User: Library Cataloguing
Date Deposited: 27 Mar 2019 08:47
Last Modified: 27 Mar 2019 08:47
URI: http://sro.sussex.ac.uk/id/eprint/82834

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