DinB is the only translesion Y family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage-inducible proteins UmuD(2) and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins, thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD(2)-interacting surface on DinB statistically covary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD(2) may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.

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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 ␤ processivity clamp ("␤") of the replicative DNA polymerase, pol III. A possible ␤-binding motif has been predicted in the same region of UmuD shown to be important for its interaction with ␤. 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 ␤-binding motif variant bind to ␤ with similar K d values as determined by changes in tryptophan fluorescence. However, these data also imply that the single tryptophan in ␤ 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.The umuDC gene products are induced as part of the SOS response and are responsible for much of the UV-induced mutagenesis in Escherichia coli (1). These gene products are subject to an elaborate set of controls that regulate their activity (1). The LexA repressor provides transcriptional control, and there are several proteolytic controls on both the umuD and umuC gene products (1). The homodimeric protein UmuD 2 is the predominant species during the first about 20 -30 min after SOS induction (2). UmuD 2 , together with UmuC, plays a role in a DNA damage checkpoint, decreasing the rate of DNA synthesis and allowing time for accurate repair processes to act (2). This correlates with the cold-sensitive phenotype observed under conditions of overexpression of the umuDC gene products (2, The wealth of structural data and models available for UmuD 2 and UmuDЈ 2 provide insight into how the two forms of the umuD gene products engage in multiple highly specific interactions ( Fig. 1) (4 -8), including with the ␣, ␤, and ⑀ subunits of the replicative polymerase, pol III (9). Of the two forms, UmuD 2 interacts more strongly with the ␤ processivity clamp (also referred to as ␤ or the ␤ clamp) than does UmuDЈ 2 (9, 10). In full-length UmuD 2 , the 39-amino acid N-terminal arms are stably bound to the globular C-terminal domain (4, 7) and form a distinct interaction surface. In t...

Products of the umuD gene in Escherichia coli play key roles in coordinating the switch from accurate DNA repair to mutagenic translesion DNA synthesis (TLS) during the SOS response to DNA damage. Homodimeric UmuD 2 is up-regulated 10-fold immediately after damage, after which slow autocleavage removes the Nterminal 24 amino acids of each UmuD. The remaining fragment, UmuD 2, is required for mutagenic TLS. The small proteins UmuD2 and UmuD 2 make a large number of specific protein-protein contacts, including three of the five known E. coli DNA polymerases, parts of the replication machinery, and RecA recombinase. We show that, despite forming stable homodimers, UmuD 2 and UmuD 2 have circular dichroism (CD) spectra with almost no ␣-helix or ␤-sheet signal at physiological concentrations in vitro. High protein concentrations, osmolytic crowding agents, and specific interactions with a partner protein can produce CD spectra that resemble the expected ␤-sheet signature. A lack of secondary structure in vitro is characteristic of intrinsically disordered proteins (IDPs), many of which act as regulators. A stable homodimer that lacks significant secondary structure is unusual but not unprecedented. Furthermore, previous single-cysteine cross-linking studies of UmuD 2 and UmuD 2 show that they have a nonrandom structure at physiologically relevant concentrations in vitro. Our results offer insights into structural characteristics of relatively poorly understood IDPs and provide a model for how the umuD gene products can regulate diverse aspects of the bacterial SOS response.natively unfolded ͉ SOS response ͉ unstructured ͉ denatured ͉ DNA repair

Animals with an open coelom do not fully constrain internal tissues, and changes in tissue or organ position during body movements cannot be readily discerned from outside of the body. This complicates modeling of soft-bodied locomotion, because it obscures potentially important changes in the center of mass as a result of internal tissue movements. We used phase-contrast synchrotron X-ray imaging and transmission light microscopy to directly visualize internal soft-tissue movements in freely crawling caterpillars. Here we report a novel visceral-locomotory piston in crawling Manduca sexta larvae, in which the gut slides forward in advance of surrounding tissues. The initiation of gut sliding is synchronous with the start of the terminal prolegs' swing phase, suggesting that the animal's center of mass advances forward during the midabdominal prolegs' stance phase and is therefore decoupled from visible translations of the body. Based on synchrotron X-ray data and transmission light microscopy results, we present evidence for a two-body mechanical system with a nonlinear elastic gut that changes size and translates between the anterior and posterior of the animal. The proposed two-body system--the container and the contained--is unlike any form of legged locomotion previously reported and represents a new feature in our emerging understanding of crawling.