ANALYSIS OF THE REGION BETWEEN AMINO-ACID-30 AND AMINO-ACID-42 OF INTACT UMUD BY A MONOCYSTEINE APPROACH

On the basis of characterizations of a set of UmuD monocysteine deriva tives, we had suggested that positions 24, 34, and 14 are closer to th e intact UmuD homodimer interface than other positions tested (M. H. L ee, T. Ohta, and G. C. Walker, J. Bacteriol, 176:4825-4837, 1994), Bec ause this region of UmuD also appeared to be important for interaction s with RecA, we followed up on our previous study by constructing a se cond set of monocysteine UmuD derivatives with single cysteine substit utions at positions 30 to 42, We found that like the VC34 mutant, UmuD derivatives with monocysteine substitutions at positions 32 and 35 sh owed deficiencies in in vivo and in vitro RecA-mediated cleavage as we ll as in UV mutagenesis, suggesting that the position 32 to 35 region mag be important for RecA-mediated cleavage of UmuD. Interestingly, Um uD with monocysteine substitutions at residues 33 and 40 showed a redu ction in UV mutagenesis while retaining the ability to be cleaved by R ecA in vivo, suggesting a deficiency in the subsequent role of the Umu D' derivatives in mutagenesis, All of the UmuD monocysteine derivative s 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 quantitativ ely upon addition of iodine and get were poorly modified with iodoacet ate led us to suggest that the pairs of residues at positions 37 and 3 8 are extremely close to the UmuD(2) homodimer interface, These observ ations indicate that the structure of the UmuDZ homodimer in solution is very different from the crystal structure of the UmuD'(2) homodimer reported by Feat 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).