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Journal of Bacteriology, January 2001, p. 347-357, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.347-357.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Genetic and Biochemical Characterization of a Novel umuD Mutation: Insights into a Mechanism for UmuD Self-Cleavage

Mark D. Sutton, Melanie Kim,dagger and Graham C. Walker*

Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 10 July 2000/Accepted 4 October 2000

Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD2 and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD2 for self-cleavage.


* Corresponding author. Mailing address: Biology Department, Massachusetts Institute of Technology, Cambridge, MA 02139. Phone: (617) 253-6716. Fax: (617) 253-2643. E-mail: gwalker{at}MIT.EDU.

dagger Present address: U.S.C. Keck School of Medicine, Los Angeles, CA 90033.


Journal of Bacteriology, January 2001, p. 347-357, Vol. 183, No. 1
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.1.347-357.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.



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