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J. Biol. Chem., Vol. 281, Issue 14, 9633-9640, April 7, 2006

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A Non-cleavable UmuD Variant That Acts as a UmuD' Mimic^*

Penny J. Beuning, Sharotka M. Simon¹, Adam Zemla, Daniel Barsky^¶, and Graham C. Walker²

From the Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the Computations Directorate and ^¶Biosciences Directorate, Lawrence Livermore National Laboratory, Livermore, California 94550

UmuD₂ cleaves and removes its N-terminal 24 amino acids to form UmuD'₂, which activates UmuC for its role in UV-induced mutagenesis in Escherichia coli. Cells with a non-cleavable UmuD exhibit essentially no UV-induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the processivity clamp ("") of the replicative DNA polymerase, pol III. A possible -binding motif has been predicted in the same region of UmuD shown to be important for its interaction with . We performed alanine-scanning mutagenesis of this motif (¹⁴TFPLF¹⁸) in UmuD and found that it has a moderate influence on UV-induced mutagenesis but is required for the cold-sensitive phenotype caused by elevated levels of wild-type UmuD and UmuC. Surprisingly, the wild-type and the -binding motif variant bind to with similar K_d values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in is in strikingly different environments in the presence of the wild-type versus the variant UmuD proteins, suggesting a distinct change in some aspect of the interaction with little change in its strength. Despite the fact that this novel UmuD variant is non-cleavable, we find that cells harboring it display phenotypes more consistent with the cleaved form UmuD', such as resistance to killing by UV light and failure to exhibit the cold-sensitive phenotype. Cross-linking and chemical modification experiments indicate that the N-terminal arms of the UmuD variant are less likely to be bound to the globular domain than those of the wild-type, which may be the mechanism by which this UmuD variant acts as a UmuD' mimic.

Received for publication, October 12, 2005 , and in revised form, January 31, 2006.

^* This work was supported in part by National Institutes of Health NCI Grant CA21615, National Institutes of Health NIEHS Center Grant P30ES02109 from the MIT Center for Environmental Health Sciences, an American Cancer Society Professorship (to G. C. W.), and a postdoctoral fellowship from the Damon Runyon Cancer Research Foundation (to P. J. B.). Some of this work was performed under the auspices of the United States Department of Energy by the University of California, Lawrence Liver-more National Laboratory Contract W-7405-Eng-48. 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. S1 and S2 and Table S1.

¹ Supported by a Cleo and Paul Schimmel Fellowship.

² To whom correspondence should be addressed: 68-633, 77 Massachusetts Ave., Cambridge, MA 02139. Tel.: 617-253-6716; Fax: 617-253-2643; E-mail: gwalker{at}mit.edu.

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