Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents

This Article Full Text Full Text (PDF, 5176K) PDF+supp data (7333K) PPT slides of all figures Supplemental Material Alert me when this article is cited Citation Map Services Email this article Similar articles in this journal Similar articles in PubMed Alert me to new content in the JCB Download to citation manager Citing Articles Citing Articles via CrossRef Google Scholar Articles by Smith, K. D. Articles by Brown, E. J. PubMed PubMed Citation Articles by Smith, K. D. Articles by Brown, E. J. Social Bookmarking                            What's this?

Tim–Tipin dysfunction creates an indispensible reliance on the ATR–Chk1 pathway for continued DNA synthesis

¹ Abramson Family Cancer Research Institute and ² Department of Cancer Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

Correspondence to Eric J. Brown: brownej{at}mail.med.upenn.edu

The Tim (Timeless)–Tipin complex has been proposed to maintain genome stability by facilitating ATR-mediated Chk1 activation. However, as a replisome component, Tim–Tipin has also been suggested to couple DNA unwinding to synthesis, an activity expected to suppress single-stranded DNA (ssDNA) accumulation and limit ATR–Chk1 pathway engagement. We now demonstrate that Tim–Tipin depletion is sufficient to increase ssDNA accumulation at replication forks and stimulate ATR activity during otherwise unperturbed DNA replication. Notably, suppression of the ATR–Chk1 pathway in Tim–Tipin-deficient cells completely abrogates nucleotide incorporation in S phase, indicating that the ATR-dependent response to Tim–Tipin depletion is indispensible for continued DNA synthesis. Replication failure in ATR/Tim-deficient cells is strongly associated with synergistic increases in H2AX phosphorylation and DNA double-strand breaks, suggesting that ATR pathway activation preserves fork stability in instances of Tim–Tipin dysfunction. Together, these experiments indicate that the Tim–Tipin complex stabilizes replication forks both by preventing the accumulation of ssDNA upstream of ATR–Chk1 function and by facilitating phosphorylation of Chk1 by ATR.

Abbreviations used in this paper: 4-OHT, 4-hydroxytamoxifen; DSB, double-strand break; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IR, ionizing radiation; PFGE, pulsed field gel electrophoresis; PI, propidium iodide; RPA, replication protein A; shRNA, short hairpin RNA; ssDNA, single-stranded DNA.

© 2009 Smith et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Facebook

Reddit

Technorati

Twitter

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents