ROLE OF SPECIFIC AMINO-ACID-RESIDUES IN T4 ENDONUCLEASE-V THAT ALTER NONTARGET DNA-BINDING

Endonuclease V is a pyrimidine dimer-specific DNA glycosylase-apurinic (AP)(1) lyase which, in viva or at low salt concentrations in vitro, binds nontarget DNA through electrostatic interactions and remains ass ociated with that DNA until all dimers have been recognized and incise d. On the basis of the analyses of previous mutants that effect this p rocessive nicking activity, and the recently published cocrystal struc ture of a catalytically deficient endonuclease V with pyrimidine dimer -containing DNA [Vassylyev, D. G., ct al. (1995) Cell 83, 773-782], fo ur site-directed mutations were created, the mutant enzymes expressed in repair-deficient Escherichia coli, and the enzymes purified to homo geneity. Steady-state kinetic analyses revealed that one of the mutant s, Q15R, maintained an efficiency (k(cat)/K-m) near that of the wild-t ype enzyme, while R117N and K86N had a 5-10-fold reduction in efficien cy and K121N was reduced almost 100-fold. In addition, K121N and K86N exhibited a 3-5-fold increase in K-m, respectively. All the mutants ex perienced mild to severe reduction in catalytic activity (k(cat)), wit h K121N being the most severely affected (35-fold reduction). Two of t he mutants, K86N and K121N, showed dramatic effects in their ability t o scan nontarget DNA and processively incise at pyrimidine dimers in W -irradiated DNA. These enzymes (K86N and K121N) appeared to utilize a distributive, three-dimensional search mechanism even at low salt conc entrations. Q15R and R117N displayed somewhat diminished processive ni cking activities relative to that of the wild-type enzyme. These resul ts, combined with previous analyses of other mutant enzymes and the co crystal structure, provide a detailed architecture of endonuclease V-n ontarget DNA interactions.