MCB Accepts, published online ahead of print on 12 February 2007
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Mol. Cell. Biol. doi:10.1128/MCB.02190-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The human Tim/Tipin complex coordinates an intra-S checkpoint response to UV that slows replication fork displacement

Keziban Ünsal-Kaçmaz, Paul D. Chastain, Ping-Ping Qu, Parviz Minoo, Marila Cordeiro-Stone, Aziz Sancar, and William K. Kaufmann*

Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; Department of Biostatistics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; School of Medicine, University of Southern California, Los Angeles, California 90033; Center for Environmental Health and Susceptibility and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599

* To whom correspondence should be addressed. Email: wkarlk{at}med.unc.edu.


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Abstract

UV-induced DNA damage stalls DNA replication forks and activates the intra-S checkpoint to inhibit replicon initiation. In response to stalled replication forks ATR phosphorylates and activates the transducer kinase Chk1 through interactions with the mediator proteins TopBP1, Claspin and Timeless (Tim). Murine Tim recently was shown to form a complex with Timeless-interacting protein (Tipin) and a similar complex was shown here to exist in human cells. Knockdown of Tipin using siRNA reduced expression of Tim and reversed the intra-S checkpoint response to UVC. Tipin interacted with RPA and RPA-coated DNA, and RPA promoted the loading of Tipin onto RPA-free DNA. Immuno-fluorescence analysis of spread DNA fibers showed that treating HeLa cells with 2.5 J/m2 UVC not only inhibited the initiation of new replicons but also reduced the rate of chain elongation at active replication forks. Depletion of Tim and Tipin reversed the UV-induced inhibition of replicon initiation but affected the rate of DNA synthesis at replication forks in different ways. In undamaged cells depleted of Tim, the apparent rate of replication fork progression was 52% of control. In contrast, Tipin depletion had little or no effect on fork progression in un-irradiated cells but significantly attenuated the UV-induced inhibition of DNA chain elongation. Together, these findings indicate that the Tim-Tipin complex mediates the UV-induced intra-S checkpoint, Tim is needed to maintain DNA replication fork movement in the absence of damage, Tipin interacts with RPA on DNA, and in UV-damaged cells Tipin slows DNA chain elongation in active replicons.




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