Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage.

When an organism's DNA replication machinery encounters a lesion in the DNA that, for a variety of reasons, was not repaired by accurate repair pathways, it stalls, leading to one of two possible outcomes: (i) damage avoidance, a poorly understood set of processes, including daughter strand gap repair, that appear to utilize the information in the newly synthesized daughter strand of the DNA duplex to somehow bypass the lesion (17) or (ii) translesion synthesis (TLS), in which a specialized DNA polymerase is recruited for bypassing the damaged site (17, 21, 67, 69). The latter pathway is potentially mutagenic due to the miscoding or noncoding nature of the DNA lesion (17). TLS in Escherichia coli is dependent on the umuDC and recA gene products (17, 23, 61). The umuDC operon encodes a DNA polymerase, DNA polymerase V, with the unique ability to replicate over particular types of DNA lesions, including abasic sites and thymine-thymine cyclobutane dimers (55, 64, 65). Recent work indicates that UmuC is the founding member of a diverse and ubiquitous family of DNA polymerases capable of copying imperfect templates (16, 18, 21, 69). In addition to their role in TLS, the umuDC gene products also participate in cell cycle checkpoint control (41, 46). In response to DNA damage, RecA protein nucleates on single-stranded DNA (ssDNA) that is generated by the cell's failed attempts to bypass lesions in its genome (17, 57). These RecA-ssDNA nucleoprotein filaments act to mediate the cleavage of the LexA repressor (33, 34). Cleavage of LexA inactivates it as a repressor, leading to the expression of the SOS regulon, a collection of ∼30 unlinked genes whose expression is coordinately regulated (11, 17, 25). The umuDC operon is among these ∼30 LexA-regulated genes (9, 17). Importantly, UmuD also undergoes RecA-ssDNA-mediated cleavage. This cleavage serves to remove its amino-terminal 24 residues to produce UmuD′ (5, 43, 58). It has been proposed that cleavage of UmuD serves to regulate the two physiological roles of the umuDC gene products so that they act in a temporally ordered fashion (41, 46), first by participating in a DNA damage checkpoint control system and second by participating in TLS. UmuD is related to three distinct classes of proteins: (i) other UmuD-like proteins involved in mutagenesis, which also undergo RecA-ssDNA-facilitated cleavage (17, 29, 30, 52), (ii) transcriptional repressors belonging to the LexA-like family, which undergo RecA-ssDNA-facilitated cleavage (17, 52), and (iii) signal peptidases (49). Interestingly, despite the fact that UmuD′ and E. coli signal peptidase have little sequence identity apart from their lysine-serine dyad, they have a remarkable degree of structural identity. Comparison of their crystal structures revealed that 69 C-α atoms of UmuD′ are superimposable (with a root mean squared of 1.6 Å) on the E. coli signal peptidase crystal structure (49). The E. coli UmuD and UmuD′ proteins interact with each other to form homo- and heterodimers (1, 5, 22) and also interact with UmuC (4, 22, 70), RecA-ssDNA nucleoprotein filaments (5, 32), and three components of the replicative DNA polymerase (62). Given the rather small sizes of UmuD and UmuD′, an important question is which part(s) of UmuD and UmuD′ is involved in interaction with each of these other proteins? A detailed understanding of these interactions will be required for a complete understanding of the molecular mechanisms underlying the roles of the umuDC gene products in checkpoint control and TLS. Determination of the crystal (10, 50) and solution (A. E. Ferentz, G. C. Walker, and G. Wagner, unpublished data) structures of the UmuD′2 homodimer and genetic studies (39, 45) have identified the residues involved in its dimerization interface. Furthermore, biochemical characterizations of single-cysteine derivatives of UmuD and UmuD′ by analyzing homo- and heterodimer cross-linking efficiency using thiol-specific cross-linking reagents and by studies of spin-labeled derivatives by electron spin resonance have identified important components of the dimerization interfaces of the UmuD-UmuD′ heterodimer and the UmuD2 homodimer (unpublished data). Finally, these single-cysteine derivatives of UmuD have also been used, in conjunction with the thiol-specific, photoactivatable, heterobifunctional cross-linking agent p-azidoiodoacetanilide (71), to identify residues of UmuD able to cross-link efficiently to RecA-ssDNA nucleoprotein filaments (32) (Fig. ​(Fig.1).1). Taken together, these studies have begun to provide a detailed molecular understanding of the roles of the umuD gene products in regulation of the cell cycle and TLS. FIG. 1 Side (A) and top (B) views of the UmuD′2 homodimer crystal structure reported by Peat et al. (50) with the homodimer interface reported by Ferentz et al. (10). Leu101 and Arg102 are shown in red. Val34, Ser57, Ser67, Ser81, and Ser112 are shown ... As part of our ongoing effort to better understand how UmuD and UmuD′ interact with other proteins to enable checkpoint control and TLS, we have embarked on a site-directed mutational analysis of the umuD gene products (1, 45). Comparison of the deduced amino acid sequences of members of the UmuD-like mutagenesis proteins to those of the LexA-like transcriptional repressor family identified a small number of residues that were well conserved exclusively among the UmuD-like mutagenesis proteins (1) (see Fig. ​Fig.2).2). This observation suggested that these might be residues that are important for the biological roles of the umuD gene products rather than for the RecA-ssDNA-facilitated cleavage of UmuD. Mutations affecting most of these highly conserved positions have already been characterized (19, 39, 45). Here we describe our construction and genetic and biochemical characterizations of a UmuD derivative in which two such highly conserved residues, leucine-101 (Leu101) and arginine-102 (Arg102), have been replaced with glycines. Our characterizations of this mutant UmuD protein, which we unexpectedly found to be deficient in RecA-ssDNA-facilitated cleavage, suggest a possible mechanism for how the interaction of UmuD2 with the RecA-ssDNA nucleoprotein filament results in the cleavage of one UmuD molecule by its intradimer partner to yield UmuD′ (10, 37). FIG. 2 Partial amino acid alignment of proteins similar to E. coli UmuD. Shown is the region between amino acids 94 and 108 of UmuD. This figure is modified from references 1 and 51. UmuD-like mutagenesis proteins (I) and LexA-like transcriptional repressors ...