RNF8-independent Lys63 poly-ubiquitylation prevents genomic instability in response to replication-associated DNA damage

Chantal H M A Ramaekers; Twan van den Beucken; Robert G Bristow; Roland K Chiu; Daniel Durocher; Bradly G Wouters

doi:10.1371/journal.pone.0089997

RNF8-independent Lys63 poly-ubiquitylation prevents genomic instability in response to replication-associated DNA damage

PLoS One. 2014 Feb 28;9(2):e89997. doi: 10.1371/journal.pone.0089997. eCollection 2014.

Authors

Chantal H M A Ramaekers¹, Twan van den Beucken¹, Robert G Bristow², Roland K Chiu³, Daniel Durocher⁴, Bradly G Wouters⁵

Affiliations

¹ Ontario Cancer Institute and Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada ; Maastricht Radiation Oncology (MaastRO) Lab, GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands.
² Ontario Cancer Institute and Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada ; Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.
³ Department of Radiation Oncology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.
⁴ Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada ; Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.
⁵ Ontario Cancer Institute and Campbell Family Institute for Cancer Research, Princess Margaret Cancer Centre, University Health Network, Toronto, Ontario, Canada ; Maastricht Radiation Oncology (MaastRO) Lab, GROW - School for Oncology and Developmental Biology, Maastricht University, Maastricht, The Netherlands ; Departments of Radiation Oncology and Medical Biophysics, University of Toronto, Toronto, Ontario, Canada.

Abstract

The cellular response to DNA double strand breaks (DSBs) involves the ordered assembly of repair proteins at or near sites of damage. This process is mediated through post-translational protein modifications that include both phosphorylation and ubiquitylation. Recent data have demonstrated that recruitment of the repair proteins BRCA1, 53BP1, and RAD18 to ionizing irradiation (IR) induced DSBs is dependent on formation of non-canonical K63-linked polyubiquitin chains by the RNF8 and RNF168 ubiquitin ligases. Here we report a novel role for K63-ubiquitylation in response to replication-associated DSBs that contributes to both cell survival and maintenance of genome stability. Suppression of K63-ubiquitylation markedly increases large-scale mutations and chromosomal aberrations in response to endogenous or exogenous replication-associated DSBs. These effects are associated with an S-phase specific defect in DNA repair as revealed by an increase in residual 53BP1 foci. Use of both knockdown and knockout cell lines indicates that unlike the case for IR-induced DSBs, the requirement for K63-ubiquitylation for the repair of replication associated DSBs was found to be RNF8-independent. Our findings reveal the existence of a novel K63-ubiquitylation dependent repair pathway that contributes to the maintenance of genome integrity in response to replication-associated DSBs.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

BRCA1 Protein / genetics
BRCA1 Protein / metabolism
Cell Line, Tumor
DNA Breaks, Double-Stranded / radiation effects*
DNA Repair*
DNA Replication*
DNA-Binding Proteins / genetics
DNA-Binding Proteins / metabolism
Epithelial Cells / cytology
Epithelial Cells / metabolism
Epithelial Cells / radiation effects
Gene Expression Regulation
Genomic Instability*
Humans
Intracellular Signaling Peptides and Proteins / genetics
Intracellular Signaling Peptides and Proteins / metabolism
Lysine / metabolism
Phosphorylation
Protein Processing, Post-Translational*
Radiation, Ionizing
Signal Transduction
Tumor Suppressor p53-Binding Protein 1
Ubiquitin-Protein Ligases
Ubiquitination

Substances

BRCA1 Protein
BRCA1 protein, human
DNA-Binding Proteins
Intracellular Signaling Peptides and Proteins
RAD18 protein, human
RNF8 protein, human
TP53BP1 protein, human
Tumor Suppressor p53-Binding Protein 1
Ubiquitin-Protein Ligases
Lysine

Grants and funding

This work was financially supported by the Transnational University Limburg (30943045T to BW), the Dutch Cancer Society, the Ontario Ministry of Health and Long Term Care (OMOHLTC), the Ontario Institute for Cancer Research, and the Terry Fox Research Institute (Selective therapies program to BW). The views expressed do not necessarily reflect those of the OMOHLTC. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.