Replication of O6-methyl-dG in Saccharomyces cerevisiae Público

Abstract

This study investigated the mechanisms used to replicate across O⁶-methyl-dG (^MeG) in Saccharomyces cerevisiae. Single stranded oligonucleotide transformation was used to incorporate one ^MeG lesion into the yeast genome. By introducing the lesion into several different strain backgrounds and examining the sequences after replication, this study explored the roles of mismatch repair (MMR), translesion synthesis (TLS), and template switching in accurately replicating across the ^MeG lesion. In contrast to previous results, this study found that MMR is crucial to the accurate replication of ^MeG. TLS also seems to contribute to the replication of the ^MeG lesion, particularly when the lesion is in the lagging strand. However, this replication via TLS is typically inaccurate in the absence of MMR. Finally, this study discovered that Rad5, which is typically associated with template switching, plays a significant role in bypassing the lesion and promoting accurate replication across the lesion by influencing other mechanisms, like TLS.

Table of Contents

Abstract...1

Introduction...1

Alkylation damage...2

O6-methyl-dG (MeG)...2

Methods for coping with MeG in yeast...3

Oligonucleotide transformation in yeast...6

Experiments in this study...7

Methods...8

Yeast strains...8

Oligonucleotides used...9

Single-stranded oligonucleotide transformation...10

SphI restriction site test...11

Sequencing...12

Results...13

Oligo transformation...13

SphI restriction site test...15

Sequencing data...16

Discussion...17

Oligo incorporation...17

Replication of the MeG lesion...18

Conclusions and future experiments...22

List of Figures...26

List of Tables...40

References...44

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	BS
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics
Palabra Clave	translesion synthesis O6-methyl-dG yeast mismatch repair alkylation damage template switching Saccharomyces cerevisiae
Committee Chair / Thesis Advisor	Crouse, Gray, Emory University
Committee Members	Spell, Rachelle, Emory University Kow, Yoke Wah, Emory University

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