Abstract
T4 endonuclease V catalyzes the hydrolysis of the glycosyl bond of a thymine dimer in a DNA duplex and the cleavage of the 3'-phosphate by beta-elimination. We have previously identified a catalytic site for the first reaction (pyrimidine dimer-glycosylase activity) by systematic mutagenesis (Doi et al. Proc. Natl. Acad. Sci. USA 1992 in press) and by x-ray crystallography (Morikawa et al. Science, 256: 523-526, 1992). The results showed that replacement of Glu23 with either glutamine or aspartic acid completely abolished the glycosylase activity. We describe the investigation of the second reaction (apurinic/apyrimidinic endonuclease activity), using twenty two mutants of T4 endonuclease V plus a DNA mini duplex containing an abasic site. Replacement of Glu23 by glutamine abolished the second reaction, but replacement with aspartic acid did not. The pH optima of the mutant (23 Asp) and the wild type were found to be 5.0 and 5.5, respectively. We conclude that the carboxylate anion in position 23 may act as a general base in the beta-elimination reaction of the endonuclease.
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- Bailly V., Verly W. G. Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst. Biochem J. 1987 Mar 1;242(2):565–572. doi: 10.1042/bj2420565. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bertrand J. R., Vasseur J. J., Rayner B., Imbach J. L., Paoletti J., Paoletti C., Malvy C. Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site. Nucleic Acids Res. 1989 Dec 25;17(24):10307–10319. doi: 10.1093/nar/17.24.10307. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dowd D. R., Lloyd R. S. Site-directed mutagenesis of the T4 endonuclease V gene: the role of arginine-3 in the target search. Biochemistry. 1989 Oct 31;28(22):8699–8705. doi: 10.1021/bi00448a005. [DOI] [PubMed] [Google Scholar]
- Inaoka T., Ishida M., Ohtsuka E. Affinity of single- or double-stranded oligodeoxyribonucleotides containing a thymine photodimer for T4 endonuclease V. J Biol Chem. 1989 Feb 15;264(5):2609–2614. [PubMed] [Google Scholar]
- Mazumder A., Gerlt J. A., Absalon M. J., Stubbe J., Cunningham R. P., Withka J., Bolton P. H. Stereochemical studies of the beta-elimination reactions at aldehydic abasic sites in DNA: endonuclease III from Escherichia coli, sodium hydroxide, and Lys-Trp-Lys. Biochemistry. 1991 Jan 29;30(4):1119–1126. doi: 10.1021/bi00218a033. [DOI] [PubMed] [Google Scholar]
- Morikawa K., Matsumoto O., Tsujimoto M., Katayanagi K., Ariyoshi M., Doi T., Ikehara M., Inaoka T., Ohtsuka E. X-ray structure of T4 endonuclease V: an excision repair enzyme specific for a pyrimidine dimer. Science. 1992 Apr 24;256(5056):523–526. doi: 10.1126/science.1575827. [DOI] [PubMed] [Google Scholar]
- Nakabeppu Y., Sekiguchi M. Physical association of pyrimidine dimer DNA glycosylase and apurinic/apyrimidinic DNA endonuclease essential for repair of ultraviolet-damaged DNA. Proc Natl Acad Sci U S A. 1981 May;78(5):2742–2746. doi: 10.1073/pnas.78.5.2742. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sancar A., Sancar G. B. DNA repair enzymes. Annu Rev Biochem. 1988;57:29–67. doi: 10.1146/annurev.bi.57.070188.000333. [DOI] [PubMed] [Google Scholar]
- Valerie K., Henderson E. E., deRiel J. K. Identification, physical map location and sequence of the denV gene from bacteriophage T4. Nucleic Acids Res. 1984 Nov 12;12(21):8085–8096. doi: 10.1093/nar/12.21.8085. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yasuda S., Sekiguchi M. T4 endonuclease involved in repair of DNA. Proc Natl Acad Sci U S A. 1970 Dec;67(4):1839–1845. doi: 10.1073/pnas.67.4.1839. [DOI] [PMC free article] [PubMed] [Google Scholar]