A Non-cleavable UmuD Variant That Acts as a UmuD′ Mimic

Penny J Beuning,Sharotka M Simon,Adam Zemla,Daniel Barsky,Graham C Walker

doi:10.1074/jbc.m511101200

Abstract

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.

Highlights

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
Plasmids expressing either the F18A UmuD variant, which is located at the top of the arm over the C-terminal globular domain, or the T14A/L17A/F18A (“UmuD-3A”) variant resulted in substantial decreases in induced mutagenesis, down to about 15–20% of the wildtype (Fig. 2)
This decrease in mutagenesis of cells harboring these variants was not accompanied by a corresponding decrease in survival after UV irradiation (Fig. 5); yet, typically, increased mutagenesis due to translesion synthesis by UmuDЈ2C is associated with increased survival after treatment with UV

Summary

Introduction

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. The umuD gene products interact with the ␤ clamp via both the N-terminal arms of UmuD2 and the globular domain of UmuD2 and UmuDЈ2 [10] This differential interaction appears to control, at least in part, whether the umuDC gene products act as part of a DNA damage checkpoint or as a translesion polymerase [9, 10]. These interactions with the ␤ clamp are of particular interest because sliding clamps play a key role in coordinating the multiple DNA polymerases present in cells [11,12,13,14,15]. Given the fact that this result was obtained utilizing only the five-amino acid motif in UmuD, and cross-linking experiments showed that the region of UmuD between residues 9 and 19 is important

Methods

Results

Conclusion