A Functional Analysis of SAMHD1 in DNA Double-Strand Break Repair Open Access

Nabeta, Geraldine Nabiryo (2017)

Permanent URL: https://etd.library.emory.edu/concern/etds/6682x4651?locale=en
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Abstract

Abstract

A Functional Analysis of SAMHD1 in DNA Double Stranded Break Repair

By Geraldine Nabiryo Nabeta

Genomic DNA is subject to exogenous or endogenous damage, which results in insults to the DNA as either single-strand or double-strand lesions. DNA double-strand breaks (DSBs) are predominantly repaired by two mechanisms, the homologous recombination(HR) and non-homologous end joining (NHEJ). The mechanism homologous recombination is of particular interest. SAMHD1 is a deoxynucleotide triphosphohydrolase (dNTPase) that restricts HIV-1 infection by depleting dNTPs necessary for reverse transcription and replication. SAMHD1 is also a protein assumed to regulate cell proliferation and survival in response to DNA damage. Although the direct mechanism through which SAMHD1 is involved in DNA repair is unclear, our laboratory has demonstrated a novel role in promoting DNA end resection, which facilitates DNA DSB repair via HR. Mutations and dysregulation of SAMHD1 have also been implicated in various cancers, although their functional significance is unknown. Taken together, these findings suggest that SAMHD1 association with cancer could be due to its role in the DNA DSB repair response, which is required to maintain genome integrity. For the following project, disease-relevant SAMHD1 mutations (DRSM) - D137N and K484T - reveal SAMHD1's role in double-strand break repair. I hypothesize that disease relevant mutations may affect SAMHD1's contribution in DNA end resection. This report serves as a preliminary functional analysis of SAMHD1 mutants.

Table of Contents

Table of Contents

INTRODUCTION 1

An Overview of DNA Double-Strand Break Repair 2

SAMHD1 6

The Clinical Relevance of SAMHD1 7

SAMHD1 and DNA Double-Strand Break Repair 11

Scope of the Thesis 15

MATERIALS AND METHODS 18

Site-Directed Mutagenesis 19

5-Bromo-2'-Deoxyuridine (BrdU) assay 19

Co-Immunoprecipitation 20

Immunofluorescence 21

RESULTS 22

BrdU as a marker for end resection 23

SAMHD1 knockdown impairs RPA70 foci 26

Establishing SAMHD1 and CtIP functional interaction 29

DISCUSSION 35

Future Directions 41

WORKS CITED 43

APPENDIX 46

Appendix I: Protocols 47

Appendix II: Plasmids and Primers

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