Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

Jun-Hyuk Choi; Laura A Lindsey-Boltz; Michael Kemp; Aaron C Mason; Marc S Wold; Aziz Sancar

doi:10.1073/pnas.1007856107

Back

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

Journal article

Open access

Peer reviewed

Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling

Jun-Hyuk Choi, Laura A Lindsey-Boltz, Michael Kemp, Aaron C Mason, Marc S Wold and Aziz Sancar

Proceedings of the National Academy of Sciences - PNAS, Vol.107(31), pp.13660-13665

From the Cover

08/03/2010

DOI: 10.1073/pnas.1007856107

PMCID: PMC2922256

PMID: 20616048

Files and links (1)

url

https://doi.org/10.1073/pnas.1007856107View

Published (Version of record) Open Access

Abstract

ATR kinase is a critical upstream regulator of the checkpoint response to various forms of DNA damage. Previous studies have shown that ATR is recruited via its binding partner ATR-interacting protein (ATRIP) to replication protein A (RPA)-covered single-stranded DNA (RPA-ssDNA) generated at sites of DNA damage where ATR is then activated by TopBP1 to phosphorylate downstream targets including the Chk1 signal transducing kinase. However, this critical feature of the human ATR-initiated DNA damage checkpoint signaling has not been demonstrated in a defined system. Here we describe an in vitro checkpoint system in which RPA-ssDNA and TopBP1 are essential for phosphorylation of Chk1 by the purified ATR-ATRIP complex. Checkpoint defective RPA mutants fail to activate ATR kinase in this system, supporting the conclusion that this system is a faithful representation of the in vivo reaction. Interestingly, we find that an alternative form of RPA (aRPA), which does not support DNA replication, can substitute for the checkpoint function of RPA in vitro, thus revealing a potential role for aRPA in the activation of ATR kinase. We also find that TopBP1 is recruited to RPA-ssDNA in a manner dependent on ATRIP and that the N terminus of TopBP1 is required for efficient recruitment and activation of ATR kinase.

Biological Sciences

DNA damage checkpoint

signal transduction

topoisomerase IIβ binding protein 1

Details

Title: Subtitle: Reconstitution of RPA-covered single-stranded DNA-activated ATR-Chk1 signaling
Creators: Jun-Hyuk Choi - Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
Laura A Lindsey-Boltz - Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
Michael Kemp - Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
Aaron C Mason - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
Marc S Wold - Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA 52242
Aziz Sancar - Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, NC 27599; and
Resource Type: Journal article
Publication Details: Proceedings of the National Academy of Sciences - PNAS, Vol.107(31), pp.13660-13665
Series: From the Cover
DOI: 10.1073/pnas.1007856107
PMID: 20616048
PMCID: PMC2922256
NLM abbreviation: Proc Natl Acad Sci U S A
ISSN: 0027-8424
eISSN: 1091-6490
Publisher: National Academy of Sciences
Language: English
Date published: 08/03/2010
Academic Unit: Radiation Oncology; Biochemistry and Molecular Biology
Record Identifier: 9984024412502771

Metrics

11 Record Views

111 Times Cited - Web of Science