Mutations affecting the ability of the Escherichia coli UmuD ' protein to participate in SOS mutagenesis

The products of the SOS-regulated umuDC operon are required for most UV and chemical mutagenesis in Escherichia call, a process that results from a tr anslesion synthesis mechanism. The UmuD protein is activated for its role i n mutagenesis by a RecA-facilitated autodigestion that removes the N-termin al 24 amino acids. A previous genetic screen for nonmutable umuD mutants ha d 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 87:719 0-7194, 1990). To identify elements of the UmuD' protein necessary for its role in translesion synthesis, we began with umuD', a modified form of the umuD gene that directly encodes the UmuD' protein, and obtained missense um uD' mutants deficient in UV and methyl methanesulfonate mutagenesis. The D3 9G, L40R, and T51I mutations affect residues located at the UmuD'(2) homodi mer interface and interfere with homodimer formation in vivo. The D75A muta tion affects a highly conserved residue located at one end of the central s trand in a three-stranded beta-sheet and appears to interfere with UmuD', h omodimer formation indirectly by affecting the structure of the UmuD' monom er, When expressed from a multicopy plasmid, the L40R umuD' mutant gene exh ibited a dominant negative effect on a chromosomal umuD(+) gene with respec t to UV mutagenesis, suggesting that the mutation has an effect on UmuD' fu nction that goes beyond its impairment of homodimer formation. The G129D mu tation affects a highly conserved residue that lies at the end of the long C-terminal beta-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 Um uD'(2) homodimer,,which are mobile in solution.