A non-cleavable UmuD variant that acts as a UmuD' mimic.
UmuD(2) 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 beta processivity clamp ("beta") of the replicative DNA polymerase, pol III. A possible beta-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with beta. We performed alanine-scanning mutagenesis of this motif ((14)TFPLF(18)) 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 beta-binding motif variant bind to beta with similar K(d) values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in beta 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.
Beuning
Penny J
PJ
Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Simon
Sharotka M
SM
Zemla
Adam
A
Barsky
Daniel
D
Walker
Graham C
GC
D000409:Alanine; D004258:DNA Polymerase III / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010641:Phenotype; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry; D020798:Two-Hybrid System Techniques; D014466:Ultraviolet Rays
D029968:Escherichia coli Proteins; D014364:Tryptophan; C078202:UmuC protein, E coli; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D000409:Alanine