A non-cleavable UmuD variant that acts as a UmuD′ mimic

Abstract

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 β processivity clamp ("β") of the replicative DNA polymerase, 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. 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 β-binding motif variant bind to β with similar Kd values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in β 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.