Mutations Affecting the Ability of the Escherichia coli UmuD′ Protein To Participate in SOS Mutagenesis

Abstract

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia coli, a process that results from a translesion synthesis mechanism. The UmuD protein is activated for its role in mutagenesis by a RecA-facilitated autodigestion that removes the N-terminal 24 amino acids. A previous genetic screen for nonmutable umuDmutants had resulted in the isolation of a set of missense mutants that produced UmuD proteins that were deficient in RecA-mediated cleavage (J. R. Battista, T. Ohta, T. Nohmi, W. Sun, and G. C. Walker, Proc. Natl. Acad. Sci. USA 87:7190–7194, 1990). To identify elements of the UmuD′ protein necessary for its role in translesion synthesis, we began with umuD′, a modified form of theumuD gene that directly encodes the UmuD′ protein, and obtained missense umuD′ mutants deficient in UV and methyl methanesulfonate mutagenesis. The D39G, L40R, and T51I mutations affect residues located at the UmuD′2 homodimer interface and interfere with homodimer formation in vivo. The D75A mutation affects a highly conserved residue located at one end of the central strand in a three-stranded β-sheet and appears to interfere with UmuD′2 homodimer formation indirectly by affecting the structure of the UmuD′ monomer. When expressed from a multicopy plasmid, the L40R umuD′ mutant gene exhibited a dominant negative effect on a chromosomal umuD + gene with respect to UV mutagenesis, suggesting that the mutation has an effect on UmuD′ function that goes beyond its impairment of homodimer formation. The G129D mutation affects a highly conserved residue that lies at the end of the long C-terminal β-strand and results in a mutant UmuD′ protein that exhibits a strongly dominant negative effect on UV mutagenesis in a umuD +strain. The A30V and E35K mutations alter residues in the N-terminal arms of the UmuD′2 homodimer, which are mobile in solution.

Specific amino acid changes enhance the anti‐recombination activity of the UmuD′C complex

“…2). With the exception of UmuD′ E35K, which was found in another study (Ohta et al ., 1999), they constitute a distinct set from the UmuD′ and UmuC mutations previously found to reduce the ability of the UmuD′C complex to promote SOS mutagenesis (Woodgate et al ., 1994; McLenigan et al ., 1998; Ohta et al ., 1999). Four out of the five UmuD′[Rin ++ ] amino acid changes, G25D, S28T, P29L and E35K, are located in the NH 2 ‐terminal arm of UmuD′ as defined by Peat et al .…”

“…Above the bars are indicated the position of the [Rin ++ ] amino acid changes described in this publication. Below the bars are indicated the position of amino acid changes previously isolated as reducing the capacity of the UmuD′C complex to promote SOS mutagenesis (Woodgate et al ., 1994; McLenigan et al ., 1998; Ohta et al ., 1999).…”

Hereditary stability and variation in evolution and development

“…Mechanistically, damaged bases and stalled replication forks are bound by RecA protein and this RecA becomes activated as a nuclease specific for several targets including the LexA repressor. Among the approximately 20 genes that are under LexA control are umu C and umu D. umu D is further processed by activated RecA and together these three proteins facilitate mutagenic synthesis (Ohta et al 1999). This mutagenic synthesis is immediately beneficial to cell survival because it allows synthesis over sites that lack normal base-pairing information.…”

Escherichia coli radD (yejH) gene: a novel function involved in radiation resistance and double‐strand break repair

“…The current recognized repertoire of Escherichia coli DNA repair genes has been compiled in screens carried out over a period of nearly four decades (Clark and Margulies, 1965;Howard-Flanders, 1968;Konrad, 1977;Volkert and Nguyen, 1984;Kolodner et al, 1985;Modrich, 1987;Mahdi and Lloyd, 1989;Ohta et al, 1999). Screens to identify genes involved in radiation resistance were part of these efforts.…”