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J. Biol. Chem., Vol. 281, Issue 43, 32081-32088, October 27, 2006

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Monoubiquitination of Proliferating Cell Nuclear Antigen Induced by Stalled Replication Requires Uncoupling of DNA Polymerase and Mini-chromosome Maintenance Helicase Activities^*

From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, California 94305-5441

Proliferating cell nuclear antigen (PCNA) is a homotrimeric, ring-shaped protein complex that functions as a processivity factor for DNA polymerases. Following genotoxic stress, PCNA is modified at a conserved site by either a single ubiquitin moiety or a polyubiquitin chain. These modifications are required to coordinate DNA damage tolerance processes with ongoing replication. The molecular mechanisms responsible for inducing PCNA ubiquitination are not well understood. Using Xenopus egg extracts, we show that ultraviolet radiation and aphidicolin treatment induce the mono- and diubiquitination of PCNA. PCNA ubiquitination is replication-dependent and coincides with activation of the ataxia telangiectasia mutated and Rad3-related (ATR)-dependent DNA damage checkpoint pathway. However, loss of ATR signaling by depletion of the ATR-interacting protein (ATRIP) or Rad1, a component of the 911 checkpoint clamp, does not impair PCNA ubiquitination. Primed single-stranded DNA generated by uncoupling of mini-chromosome maintenance helicase and DNA polymerase activities has been shown previously to be necessary for ATR activation. Here we show that PCNA ubiquitination also requires uncoupling of helicase and polymerase activities. We further demonstrate that replicating single-stranded DNA, which mimics the structure produced upon uncoupling, is sufficient to induce PCNA monoubiquitination. Our results suggest that PCNA ubiquitination and ATR activation are two independent events that occur in response to a common single-stranded DNA intermediate generated by functional uncoupling of mini-chromosome maintenance (MCM) helicase and DNA polymerase activities.

Received for publication, July 17, 2006 , and in revised form, September 7, 2006.

^* This work was supported by Grant 8GB0091 (to P. J. L.) from the California Breast Cancer Research Program and Grant GM62193 (to K. A. C.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ A Leukemia and Lymphoma Society Scholar. To whom correspondence should be addressed: Dept. of Molecular Pharmacology, Stanford University School of Medicine, 318 Campus Dr., Stanford, CA 94305-5441. Tel.: 650-498-4720; Fax: 650-725-4665; E-mail: cimprich{at}stanford.edu.

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