Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

Laura A Lindsey-Boltz,Michael G Kemp,Joyce T Reardon,Vanessa Derocco,Ravi R Iyer,Paul Modrich,Aziz Sancar

doi:10.1074/jbc.m113.542787

Abstract

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

Highlights

Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled
We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5؅ to 3؅ exonuclease activity of EXO1
We find that the nucleotide excision repair canonical 30 nucleotide gap enlarged by EXO1 is necessary and sufficient to activate ATR checkpoint signaling in the presence of the ATR co-activator TopBP1 protein

Summary

Background

Nucleotide excision repair and the ATR-mediated DNA damage checkpoint responses are genetically coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5؅ to 3؅ exonuclease activity. Several studies have strongly supported a model whereby the canonical 30-nucleotide gap generated by nucleotide excision repair is enlarged by EXO1, and the enlarged single-stranded gap (presumably occupied by RPA protein) constitutes the major signal for the ATRmediated checkpoint response outside of S phase [15,16,17]. We find that the nucleotide excision repair canonical 30 nucleotide gap enlarged by EXO1 is necessary and sufficient to activate ATR checkpoint signaling in the presence of the ATR co-activator TopBP1 protein This is the first in vitro system that couples nucleotide excision repair and the ATR-mediated DNA damage checkpoint

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION