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 vivo or at low salt concentrations in vitro, binds nontarget DNA through electrostatic interactions and remains associated with that DNA until all dimers have been recognized and incised. On the basis of the analyses of previous mutants that effect this processive nicking activity, and the recently published cocrystal structure of a catalytically deficient endonuclease V with pyrimidine dimer-containing DNA [Vassylyev, D. G., et al. (1995) Cell 83, 773-782], four site-directed mutations were created, the mutant enzymes expressed in repair-deficient Escherichia coli, and the enzymes purified to homogeneity. Steady-state kinetic analyses revealed that one of the mutants, Q15R, maintained an efficiency (k(cat)/Km) near that of the wild-type enzyme, while R117N and K86N had a 5-10-fold reduction in efficiency and K121N was reduced almost 100-fold. In addition, K121N and K86N exhibited a 3-5-fold increase in Km, respectively. All the mutants experienced mild to severe reduction in catalytic activity (k(cat)), with K121N being the most severely affected (35-fold reduction). Two of the mutants, K86N and K121N, showed dramatic effects in their ability to scan nontarget DNA and processively incise at pyrimidine dimers in UV-irradiated DNA. These enzymes (K86N and K121N) appeared to utilize a distributive, three-dimensional search mechanism even at low salt concentrations. Q15R and R117N displayed somewhat diminished processive nicking activities relative to that of the wild-type enzyme. These results, combined with previous analyses of other mutant enzymes and the cocrystal structure, provide a detailed architecture of endonuclease V-nontarget DNA interactions.
Nyaga
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The Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston 77555-1071, USA.
Dodson
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Lloyd
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D001483:Base Sequence; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D045647:DNA Glycosylases; D017931:DNA Primers; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009699:N-Glycosyl Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D011740:Pyrimidine Dimers / Q000378:metabolism; D013499:Surface Properties; D013696:Temperature; D014466:Ultraviolet Rays; D014764:Viral Proteins
D017931:DNA Primers; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D004247:DNA; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer); D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C036401:deoxyribopyrimidine endonucleosidase