INVESTIGATING THE LINK BETWEEN HOMOLOGOUS RECOMBINATION PROTEIN BRCA2 AND DNA METHYLATION
type: CreativeWork
description: Cancer cell genomes display numerous mutation patterns that subvert genome stability. DNA repair and damage response mechanisms are responsible for maintaining stability in normal cells. One such repair mechanism involves homologous recombination (HR) proteins BRCA2 which protects DNA replication intermediates from nuclease-mediated degradation. Emerging evidence demonstrates that HR proteins like BRCA2 co-evolved with the cytosine methylation machinery, suggesting a functional link between the two processes. Cytosine methylation is an epigenetic modification occurring across many lifeforms, primarily associated with the repression of gene expression. However, its influence on DNA replication and repair remains largely unknown. Here, we investigate the presence and mechanism of a functional link between the two processes. To overcome the lack of single-molecule tools capable of monitoring methylation dynamics, we developed and validated a hemimethylation-discriminating DNA fiber assay using the restriction endonuclease NhoI. By using this assay, we demonstrated that BRCA2 expression did not affect the function of DNMT1, the protein responsible for post replicative DNA methylation maintenance. We subsequently investigated whether DNMT1 influenced DNA replication and repair pathways. To this end, we employed an auxin-induced degron (AID) system for DNMT1 for its reversible degradation. Through the DNA fiber assay on this cell line, we demonstrated that the loss of cytosine methylation results in faster replication fork progression, revealing methylation to act as a rheostat for DNA replication. We then investigated if this impacted replication fork stability under replication stress conditions. Specifically, we investigated whether this impacted the nucleolytic degradation of the nascent strand to hydroxyurea, which usually occurs in HR-defective cells. Using DNA fiber assay, we demonstrated that the HU-induced nascent strand degradation conferred by the loss of BRCA2…
abstract: Cancer cell genomes display numerous mutation patterns that subvert genome stability. DNA repair and damage response mechanisms are responsible for maintaining stability in normal cells. One such repair mechanism involves homologous recombination (HR) proteins BRCA2 which protects DNA replication intermediates from nuclease-mediated degradation. Emerging evidence demonstrates that HR proteins like BRCA2 co-evolved with the cytosine methylation machinery, suggesting a functional link between the two processes. Cytosine methylation is an epigenetic modification occurring across many lifeforms, primarily associated with the repression of gene expression. However, its influence on DNA replication and repair remains largely unknown. Here, we investigate the presence and mechanism of a functional link between the two processes. To overcome the lack of single-molecule tools capable of monitoring methylation dynamics, we developed and validated a hemimethylation-discriminating DNA fiber assay using the restriction endonuclease NhoI. By using this assay, we demonstrated that BRCA2 expression did not affect the function of DNMT1, the protein responsible for post replicative DNA methylation maintenance. We subsequently investigated whether DNMT1 influenced DNA replication and repair pathways. To this end, we employed an auxin-induced degron (AID) system for DNMT1 for its reversible degradation. Through the DNA fiber assay on this cell line, we demonstrated that the loss of cytosine methylation results in faster replication fork progression, revealing methylation to act as a rheostat for DNA replication. We then investigated if this impacted replication fork stability under replication stress conditions. Specifically, we investigated whether this impacted the nucleolytic degradation of the nascent strand to hydroxyurea, which usually occurs in HR-defective cells. Using DNA fiber assay, we demonstrated that the HU-induced nascent strand degradation conferred by the loss of BRCA2…
author: PAPAGUDI VENKATRAM, RAJAGOPAL
language: eng
keywords: DNA Replication, DNA methylation, Settore BIOS-07/A - Biochimica
date published: 2025-12-17
date modified: 2026-07-27T20:19:08Z
provider: Università degli Studi di Milano
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Università degli Studi di Milano

PropertyValue
property ID: Handle
value: https://hdl.handle.net/2434/1198321