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A Non‐cleavable UmuD variant that acts as a UmuD’ mimic
Author(s) -
Beuning Penny,
Simon Sharotka M,
Zemla Adam,
Barsky Daniel,
Walker Graham C
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb55-a
Subject(s) - sos response , mutagenesis , wild type , alanine , alanine scanning , biology , genetics , phenotype , biochemistry , chemistry , microbiology and biotechnology , dna , amino acid , mutation , gene , dna damage , mutant
E. coli UmuD 2 cleaves off its N‐terminal 24 amino acids to form UmuD′ 2 , which activates UmuC for its role in UV‐mutagenesis. Cells with a non‐cleavable UmuD exhibit essentially no UV‐induced mutagenesis and are hypersensitive to killing by UV light. UmuD binds to the β processivity clamp (“β”) of DNA pol III. A possible β‐binding motif has been predicted in the same region of UmuD shown to be important for its interaction with β. Alanine‐scanning mutagenesis of this motif in UmuD ( 14 TFPLF 18 ) showed that the motif has a moderate effect 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 β‐motif variant bind to β with similar K d values, but the single tryptophan in β is in different environments in the presence of the wild‐type versus the variant UmuD proteins. Despite the fact that this UmuD variant is non‐cleavable, cells harboring it display phenotypes more consistent with the cleaved form UmuD′, such as resistance to killing by UV light and loss of 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. Supported by NCI and NIEHS (from the MIT CEHS) to G.C.W.; a Damon Runyon Cancer Research Foundation fellowship to P.J.B.; a Cleo and Paul Schimmel Fellowship to S.M.S.; and the U.S. DoE to the Univ. of California, LLNL.