Site-directed mutagenesis of the T4 endonuclease V gene: role of tyrosine-129 and -131 in pyrimidine dimer-specific binding.
T4 endonuclease V incises DNA at the sites of pyrimidine dimers through a two-step mechanism. These breakage reactions are preceded by the scanning of nontarget DNA and binding to pyrimidine dimers. In analogy to the synthetic tripeptides Lys-Trp-Lys and Lys-Tyr-Lys, which have been shown to be capable of producing single-strand scissions in DNA containing apurinic sites, endonuclease V has the amino acid sequence Trp-Tyr-Lys-Tyr-Tyr (128-132). Site-directed mutagenesis of the endonuclease V gene, denV, was performed at the Tyr-129 and at the Tyr-129 and Tyr-131 positions in order to convert the Tyr residues to nonaromatic amino acids to test their role in dimer-specific binding. The UV survival of repair-deficient (uvrA recA) Escherichia coli cells harboring the denV N-129 construction was dramatically reduced relative to wild-type denV+ cells. The survival of denV N-129,131 cells was indistinguishable from that of the parental strain lacking the denV gene. The mutant endonuclease V proteins were then characterized with regard to (1) dimer-specific nicking activity, (2) apurinic nicking activity, and (3) binding affinity to UV-irradiated DNA. Dimer-specific nicking activity and dimer-specific binding for both denV N-129 and N-129,131 were abolished, while apurinic-specific nicking was substantially retained in denV N-129,131 but was abolished in denV N-129. These results indicate that Tyr-129 and Tyr-131 positions of endonuclease V are at least important in pyrimidine dimer-specific binding and possibly nicking activity.
Stump
D G
DG
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Lloyd
R S
RS
D004278:DNA, Superhelical / Q000235:genetics / Q000528:radiation effects; D043224:Deoxyribonuclease (Pyrimidine Dimer); D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000528:radiation effects; D005796:Genes; D005814:Genes, Viral; D007700:Kinetics; D009154:Mutation; D011740:Pyrimidine Dimers / Q000378:metabolism*; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D014466:Ultraviolet Rays; D014764:Viral Proteins
D004278:DNA, Superhelical; D011740:Pyrimidine Dimers; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; C470698:endonuclease V, phage T4; D043224:Deoxyribonuclease (Pyrimidine Dimer)