The enhanced mutagenic potential of the MucAB proteins correlates with the highly efficient processing of the MucA protein

Abstract

Inducible mutagenesis in Escherichia coli requires the direct action of the chromosomally encoded UmuDC proteins or functional homologs found on certain naturally occurring plasmids. Although structurally similar, the five umu-like operons that have been characterized at the molecular level vary in their ability to enhance cellular and phage mutagenesis; of these operons, the mucAB genes from the N-group plasmid pKM101 are the most efficient at promoting mutagenesis. During the mutagenic process, UmuD is posttranslationally processed to an active form, UmuD'. To explain the more potent mutagenic efficiency of mucAB compared with that of umuDC it has been suggested that unlike UmuD, intact MucA is functional for mutagenesis. To examine this possibility, we have overproduced and purified the MucA protein. Although functionally similar to UmuD, MucA was cleaved much more rapidly both in vitro and in vivo than UmuD. In vivo, restoration of mutagenesis functions to normally nonmutable recA430, recA433, recA435, or recA730 A(umuDC)595::cat strains by either MucA+ or mutant MucA protein correlated with the appearance of the cleavage product, MucA'. These results suggest that most of the differences in mutagenic phenotype exhibited by MucAB and UmuDC correlate with the efficiency of posttranslational processing of MucA and UmuD rather than an inherent activity of the unprocessed proteins.

Substitution of mucAB or rumAB for umuDC alters the relative frequencies of the two classes of mutations induced by a site-specific T-T cyclobutane dimer and the efficiency of translesion DNA synthesis

“…Posttranslational processing of the UmuD-like proteins to their mutagenically active UmuDЈ-like species is a critical step in the mutagenic process. While both MucA and RumA are processed moderately efficiently to MucAЈ and RumAЈ (20,23), processing of UmuD to UmuDЈ is thought to be relatively inefficient (10,39). These differences can be readily seen by comparing the efficiency of bypass promoted by the native wild-type operon with that of the genetically engineered active species in an unirradiated cell (Table 3).…”

“…Under these conditions, UmuDC gave only 3% bypass whereas UmuDЈC gave 42% bypass, a 14-fold increase. In contrast, both MucAB and RumAB (which are efficiently processed and are likely to exist in their processed form even in an undamaged cell [20,23]) gave much higher levels of bypass (36 and 58%, respectively). Introduction of the genetically engineered MucAЈB and RumAЈB resulted in modest increases to 63 and 70% bypass levels, respectively.…”

“…Induced mutation frequencies are higher in strains expressing the muc and the rum operons rather than umuDC (4, 5, 23), an effect that has been ascribed in part to the greater susceptibility of MucA and RumA to proteolytic processing (20,23), although their relative ability to bind to a RecA-nucleoprotein filament also seems to be another mutagenically important phenotype (16,23). Different spectra of induced mutations have also been observed when mucAB has been substituted for umuDC (12,29).…”

“…Each of these mutagenesis operons possesses a lexArepressible promoter, is regulated as part of the SOS response, and promotes mutagenesis induced by many types of DNA damage. Similarly, each operon (umuDC, mucAB, or rumAB) can promote mutagenesis only if the UmuD-like protein is proteolytically processed via a RecA-mediated self-cleavage reaction generating the mutagenically active UmuDЈ-like polypeptides such as UmuDЈ, MucAЈ, and RumAЈ, respectively (10,20,23,29,33). Activity also requires the formation of a complex consisting of a homodimer of the processed UmuDЈ-like polypeptide and a single molecule of its cognate UmuClike partner (i.e., UmuC, MucB, and RumB, respectively) (40).…”

The Mutagenesis Protein MucB Interacts with Single Strand DNA Binding Protein and Induces a Major Conformational Change in Its Complex with Single-stranded DNA

“…The interaction between MucA and RecA as revealed in the two-hybrid system was expected based on the finding that RecA promotes the cleavage of MucA to MucAЈ (37,38), similarly to the cleavage of UmuD to UmuDЈ (7)(8)(9). Interactions between RecA and MucAЈ or UmuDЈ were proposed previously based on chemical cross-linking experiments (39).…”