Analysis of the region between amino acids 30 and 42 of intact UmuD by a monocysteine approach.
On the basis of characterizations of a set of UmuD monocysteine derivatives, we had suggested that positions 24, 34, and 44 are closer to the intact UmuD homodimer interface than other positions tested (M. H. Lee, T. Ohta, and G. C. Walker, J. Bacteriol. 176:4825-4837, 1994). Because this region of UmuD also appeared to be important for interactions with RecA, we followed up on our previous study by constructing a second set of monocysteine UmuD derivatives with single cysteine substitutions at positions 30 to 42. We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 showed deficiencies in in vivo and in vitro RecA-mediated cleavage as well as in UV mutagenesis, suggesting that the position 32 to 35 region may be important for RecA-mediated cleavage of UmuD. Interestingly, UmuD with monocysteine substitutions at residues 33 and 40 showed a reduction in UV mutagenesis while retaining the ability to be cleaved by RecA in vivo, suggesting a deficiency in the subsequent role of the UmuD' derivatives in mutagenesis. All of the UmuD monocysteine derivatives in the position 30 to 42 series purified indistinguishably from the wild-type protein. The observations that purified proteins of the UmuD derivatives RC37 and IC38 could be disulfide cross-linked quantitatively upon addition of iodine and yet were poorly modified with iodoacetate led us to suggest that the pairs of residues at positions 37 and 38 are extremely close to the UmuD2 homodimer interface. These observations indicate that the structure of the UmuD2 homodimer in solution is very different from the crystal structure of the UmuD'2 homodimer reported by Peat et al. (T. S. Peat, E. G. Frank, J. P. McDonald, A. S. Levine, R. Woodgate, and W. A. Hendrickson, Nature [London] 380:727-730, 1996).
Guzzo
A
A
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Lee
M H
MH
Oda
K
K
Walker
G C
GC
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003545:Cysteine; D004259:DNA-Directed DNA Polymerase; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D007455:Iodine / Q000378:metabolism; D007461:Iodoacetates / Q000494:pharmacology; D019807:Iodoacetic Acid; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010618:Phenanthrolines / Q000378:metabolism; D011938:Rec A Recombinases / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014466:Ultraviolet Rays
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D007461:Iodoacetates; D010618:Phenanthrolines; D007455:Iodine; D011938:Rec A Recombinases; D004259:DNA-Directed DNA Polymerase; C055218:UmuD protein, E coli; D003545:Cysteine; D004229:Dithiothreitol; C025205:1,10-phenanthroline; D019807:Iodoacetic Acid