Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis - PubMed (original) (raw)
Mutations affecting the ability of the Escherichia coli UmuD' protein to participate in SOS mutagenesis
T Ohta et al. J Bacteriol. 1999 Jan.
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 umuD mutants 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 the umuD 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 beta-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 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 UmuD'2 homodimer, which are mobile in solution.
Figures
FIG. 1
Effect of plasmids carrying _umuD_′ mutations on UV-induced (20 J/m2) reversion of _argE3_→Arg+ in an AB1157 umuD44 strain (GW3200) (A) and an isogenic umuD+ strain (AB1157) (B). UV mutability is expressed relative to the umuD44 strain carrying the _umuD_′ plasmid pGW2122 (17.8 induced Arg+ revertants/106 survivors) (A) and relative to the umuD+ strain AB1157 (8.3 induced Arg+ revertants/106 survivors) (B).
FIG. 2
Effect of plasmids carrying _umuD_′ mutations on MMS-induced reversion of _argE3_→Arg+ in the AB1157 umuD44 strain, GW3200.
FIG. 3
The in vivo levels of the UmuD′ mutants (A) and their ability to form UmuD′ homodimers (B) were measured in crude lysates by using chemiluminescence immunodetection as described in Materials and Methods. (A) In vivo levels of the UmuD′ mutants relative to that of wild-type UmuD′ in a lexA+ strain (GW3200) following a 20-J/m2 UV dose are shown. (B) The abilities of the various UmuD′ mutants to be cross-linked as homodimers in vivo by formaldehyde are shown. The UmuD′ mutant proteins were expressed constitutively because of the lexA(Def) allele in strain GW8025. The positions of the UmuD′ monomer and UmuD′2 homodimer are indicated.
FIG. 4
Worm representation of the UmuD′2 homodimer. Residues critical for UV mutagenesis are shown in red; those in pink are sites of missense mutations that caused less than a twofold reduction in UV mutagenesis.
FIG. 5
Close-up view of the dimerization interface showing interactions of side chains that may be important for dimerization.
FIG. 6
Space-filling model of the UmuD′ dimer, rotated by 90° relative to the view shown in Fig. 4, and illustrating the spatial relationship of residues S49, A50, T51, S76, and G129.
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