Genetic and biochemical characterization of a novel umuD mutation: insights into a mechanism for UmuD self-cleavage.
Most translesion DNA synthesis (TLS) in Escherichia coli is dependent upon the products of the umuDC genes, which encode a DNA polymerase, DNA polymerase V, with the unique ability to replicate over a variety of DNA lesions, including cyclobutane dimers and abasic sites. The UmuD protein is activated for its role in TLS by a RecA-single-stranded DNA (ssDNA)-facilitated self-cleavage event that serves to remove its amino-terminal 24 residues to yield UmuD'. We have used site-directed mutagenesis to construct derivatives of UmuD and UmuD' with glycines in place of leucine-101 and arginine-102. These residues are extremely well conserved among the UmuD-like proteins involved in mutagenesis but are poorly conserved among the structurally related LexA-like transcriptional repressor proteins. Based on both the crystal and solution structures of the UmuD' homodimer, these residues are part of a solvent-exposed loop. Our genetic and biochemical characterizations of these mutant UmuD and UmuD' proteins indicate that while leucine-101 and arginine-102 are critical for the RecA-ssDNA-facilitated self-cleavage of UmuD, they serve only a minimal role in enabling TLS. These results, and others, suggest that the interaction of RecA-ssDNA with leucine-101 and arginine-102, together with numerous other contacts between UmuD(2) and the RecA-ssDNA nucleoprotein filaments, serves to realign lysine-97 relative to serine-60, thereby activating UmuD(2) for self-cleavage.
Sutton
M D
MD
Biology Department, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Kim
M
M
Walker
G C
GC
D000483:Alleles; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003080:Cold Temperature; D004277:DNA, Single-Stranded / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D029968:Escherichia coli Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009698:Nucleoproteins; D011487:Protein Conformation; D011938:Rec A Recombinases / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D013014:SOS Response, Genetics / Q000235:genetics; D016415:Sequence Alignment
D001426:Bacterial Proteins; D004277:DNA, Single-Stranded; D029968:Escherichia coli Proteins; D009698:Nucleoproteins; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli