Elucidating the role of CHD5 domains in the recruitment of CHD5 to DNA double-strand break sites Public

Abstract

DNA double-strand breaks (DSBs) are highly deleterious and pose a threat to genome integrity when left unrepaired, potentially driving the development or progression of cancer. Non-homologous end joining (NHEJ) and homologous recombination (HR) are the two primary repair pathways that eukaryotic cells rely on to repair DSBs. However, NHEJ is more mutagenic, so maintaining the balance between the two pathways within a cell is also essential for genomic stability. The chromatin remodeler protein CHD5 has been found to play a role in the DNA damage response (DDR), which may include mediating the balance between NHEJ and HR, as unpublished results from our lab have shown that loss of CHD5 shifts the DNA repair choice toward NHEJ. Therefore, we aim to further understand the mechanics behind CHD5’s recruitment to DSBs. In this study, we investigate which domains of CHD5 are responsible for its recruitment. Using chromatin immunoprecipitation (ChIP) and real-time quantitative PCR (qPCR), we show that all domains except the domain of unknown function (DUF) and the CHDCT2 uncharacterized domain recruit independently to DSBs, suggesting that all of the domains except the DUF and CHDCT2 participate in facilitating recruitment. However, the lysine-rich domain at CHD5’s N-terminal showed the greatest enrichment at DSBs, indicating that it may play the biggest role in facilitating recruitment. Finally, we use laser microirradiation and live-cell imaging to explore the recruitment and retention kinetics of the CHD5 protein, demonstrating that CHD5 recruits rapidly and retains for an extended period of time.

Table of Contents

Introduction...1                                                                                                                

DNA double-strand break repair...1                                                                                

CHD5: A chromatin remodeler involved in cancer...1                                                    

Approaches for investigating protein recruitment to DSBs...6                                        

Hypothesis...10                                                                                                                 

Materials and Methods...11                                                                                                 

Cell Lines...11                                                                                                                  

Plasmid Extraction and Purification...11                                                                        

Transfections...11                                                                                                             

Laser Microirradiation...12                                                                                              

ImageJ Quantification...12                                                                                               

ChIP Assays...13                                                                                                             

Real-Time Quantitative PCR...14                                                                                   

Protein Lysate Preparation and Western Blotting...15                                                    

Statistical Analysis...16                                                                                                   

Results...18                                                                                                                             

Confirmation of plasmid expression...18                                                                        

Quantifying recruitment kinetics of CHD5...20                                                              

ChIP reveals GFP-CHD5 1-337 construct has the highest enrichment at

double-strand breaks...22                                                                                                

Discussion...26                                                                                                                       

Supplementary Figures...29                                                                                                  

References...31                                                                                                                       

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Molecular Biology, General Biology, Genetics
Mot-clé	DNA damage CHD5 DNA double-strand breaks Chromatin remodelers Cancer DNA repair
Committee Chair / Thesis Advisor	David S. Yu, Emory University
Committee Members	Alexandre Orthwein, Emory University Heather Skye Comstra, Emory University Edward Nam, Emory University

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