Investigating the Role of CHD5 in Replication Stress Response Restricted; Files Only

Abstract

DNA replication is a dynamic process required for genetic transmission and maintenance of genome stability. Replication stress is one of the main sources of genomic instability and a defining hallmark of cancer. When exogenous stress agents induce single stranded breaks (SSBs), cells preserve genome integrity by activating the ATR-CHK1 replication stress response (RSR) pathway, which stabilizes stalled replication forks and enforces S phase checkpoint signaling. While previous studies show that chromatin remodelers contribute to coordinating replication machinery and DNA damage response, the specific role of chromodomain helicase DNA-binding protein 5 (CHD5) in the RSR is unknown. We investigate the mechanistic role of CHD5 in RSR using multiple cell lines: H460 (non-small cell lung carcinoma), PC3 (prostate adenocarcinoma), U2OS (human bone osteosarcoma), and HEK293T (human embryonic kidney). Mass spectrometry analysis revealed CHD5 interacts with DNA replication machinery proteins and the DNA damage response pathway, implying a connection to the RSR. CHD5-depleted H460 cells demonstrated endogenous genomic instability, measured through increased spontaneous 53BP1 nuclear bodies. Cells without CHD5 were increasingly sensitized to hydroxyurea (HU)-induced replication stress in clonogenic survival assays, highlighting CHD5’s significance in maintaining genome integrity. Proximity ligation assay showed CHD5’s dynamic recruitment to active replication forks under HU-induced replication stress, enriched in S phase cells. With CHD5 placed near replication forks, we saw CHD5 depleted cells had reduced Replication protein A (RPA70) foci formation at stalled forks and impaired ATR-CHK1 checkpoint axis activation following HU treatment. These phenotypes were consistent across p53 wild type and null cell lines, suggesting CHD5’s role the RSR is independent of p53 status.

Collectively, our findings demonstrate CHD5 as a potential regulator in the early stages of the RSR signaling cascade, required for RPA loading on SSBs, ATR-CHK1 axis activation, and restoration of replication fork stability. We establish a mechanistic link between CHD5 protein and the RSR, providing insight into how CHD5 loss can contribute to genomic instability and its potential role as a prognostic marker for replication stress inducing therapeutics.

Table of Contents

Chapter 1: INTRODUCTION

1.1 DNA Replication and Genome Maintenance

1.2 Replication Stress

1.3 Replication Stress Response (RSR)

1.4 Chromatin Remodelers and CHD5

1.5 Preliminary Data Supporting CHD5’s Role in RSR

1.6 Research Objectives

 

Chapter 2: MATERIALS AND METHODS

2.1 Materials

2.2 Cell Culture and Maintenance

2.3 siRNA-Mediated Depletion of CHD5

2.4 Immunofluorescence Staining (IF)

2.5 Proximity Ligation Assay (PLA)

2.6 Western Blot Analysis

2.7 Clonogenic Survival Assay

2.8 In Situ Analysis of Protein Interactions at DNA Replication Forks (SIRF)

2.9 CRISPR/Cas9 Knockout

 

Chapter 3: RESULTS

3.1 Mass Spectrometry Analysis Indicates CHD5 Interacts with Proteins Involved in Replication Machinery in H460 Cells 

3.2 CHD5 Knockdown Validation Across Multiple Cell Lines

3.3 53BP1 Nuclear Bodies Show Increased Genomic Instability with CHD5 Depletion

3.4 CHD5 Localizes to Replication Forks Under HU-Induced Replication Stress

3.5 CHD5 Localization at Replication Forks Increase in S Phase Cells

3.6 Loss of CHD5 Sensitizes Cells to HU-Induced Replication Stress

3.7 CHD5 depletion reduces RPA70 foci formation under replication stress

3.8 CHD5 Depletion Impairs ATR-CHK1 Checkpoint Signaling Under HU-Induced Replication Stress

 

Chapter 4: DISCUSSION

4.1 Proposed Model: CHD5 is a Chromatin Gatekeeper at Stalled Replication Forks

4.2 CHD5’s Role in RSR is Independent of p53 Status

4.3 Total ATR and Total ATM Protein Level Variability

4.4 CHD5’s Potential Use as Prognostic Marker for Replication Stress Inducing Therapeutics

4.5 Limitations

4.6 Future Directions

 

REFERENCES

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Biology, Molecular
Keyword	DNA replication Replication stress response Chromatin remodelers
Committee Chair / Thesis Advisor	Eric Battaglioli, Emory University
Committee Members	David Yu, Emory University Richard Himes, Emory University

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