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Journal of Bacteriology, October 2009, p. 5910-5920, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00292-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Characterization of Novel Alleles of the Escherichia coli umuDC Genes Identifies Additional Interaction Sites of UmuC with the Beta Clamp{triangledown}

Penny J. Beuning,1,2,3* Sarah Chan,3,{dagger} Lauren S. Waters,3,{ddagger} Haripriya Addepalli,1,§ Jaylene N. Ollivierre,1 and Graham C. Walker3

Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115,1 Center for Interdisciplinary Research on Complex Systems, Northeastern University, Boston, Massachusetts 02115,2 Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 021393

Received 4 March 2009/ Accepted 7 July 2009

Translesion synthesis is a DNA damage tolerance mechanism by which damaged DNA in a cell can be replicated by specialized DNA polymerases without being repaired. The Escherichia coli umuDC gene products, UmuC and the cleaved form of UmuD, UmuD', comprise a specialized, potentially mutagenic translesion DNA polymerase, polymerase V (UmuD'2C). The full-length UmuD protein, together with UmuC, plays a role in a primitive DNA damage checkpoint by decreasing the rate of DNA synthesis. It has been proposed that the checkpoint is manifested as a cold-sensitive phenotype that is observed when the umuDC gene products are overexpressed. Elevated levels of the beta processivity clamp along with elevated levels of the umuDC gene products, UmuD'C, exacerbate the cold-sensitive phenotype. We used this observation as the basis for genetic selection to identify two alleles of umuD' and seven alleles of umuC that do not exacerbate the cold-sensitive phenotype when they are present in cells with elevated levels of the beta clamp. The variants were characterized to determine their abilities to confer the umuD'C-specific phenotype UV-induced mutagenesis. The umuD variants were assayed to determine their proficiencies in UmuD cleavage, and one variant (G129S) rendered UmuD noncleaveable. We found at least two UmuC residues, T243 and L389, that may further define the beta binding region on UmuC. We also identified UmuC S31, which is predicted to bind to the template nucleotide, as a residue that is important for UV-induced mutagenesis.

* Corresponding author. Mailing address: Department of Chemistry and Chemical Biology, Northeastern University, 360 Huntington Ave., 102 Hurtig Hall, Boston, MA 02115. Phone: (617) 373-2865. Fax: (617) 373-8795. E-mail: beuning{at}neu.edu

{triangledown} Published ahead of print on 24 July 2009.

{dagger} Present address: Division of Urology, University of Maryland Medical Center, Baltimore, MD 21201.

{ddagger} Present address: NIH, CBMB/NICHD, Building 18, Room 113, 18 Library Drive, MSC 5430, Bethesda, MD 20892-5430.

§ Present address: Alnylam Pharmaceuticals, Cambridge, MA 02142.

Journal of Bacteriology, October 2009, p. 5910-5920, Vol. 191, No. 19
0021-9193/09/$08.00+0     doi:10.1128/JB.00292-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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