Abstract
The interaction between endonuclease V, the cyclobutane pyrimidine dimer-specific N-glycosylase/abasic lyase from bacteriophage T4, and DNA was investigated by DNase I footprinting methods. The catalytically inactive mutant E23Q was found to interact with a smaller region of DNA at the abasic site analog, tetrahydrofuran, than at a thymine dimer site. Like the wild-type enzyme, the mutant contacted the DNA substrates primarily on the strand opposite the damage. The various complexes examined by footprinting techniques represent distinct points along the catalytic pathway of endonuclease V: before catalysis at a dimer, after N-glycosylase action but before abasic lyase action, and before catalysis at an abasic site. The differences between the footprints of the mutant and wild-type enzymes on both DNA substrates likely represent subtly different conformations within these complexes.
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Selected References
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- Baer M., Sancar G. B. Photolyases from Saccharomyces cerevisiae and Escherichia coli recognize common binding determinants in DNA containing pyrimidine dimers. Mol Cell Biol. 1989 Nov;9(11):4777–4788. doi: 10.1128/mcb.9.11.4777. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bailly V., Sente B., Verly W. G. Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts. Biochem J. 1989 May 1;259(3):751–759. doi: 10.1042/bj2590751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Dodson M. L., Schrock R. D., 3rd, Lloyd R. S. Evidence for an imino intermediate in the T4 endonuclease V reaction. Biochemistry. 1993 Aug 17;32(32):8284–8290. doi: 10.1021/bi00083a032. [DOI] [PubMed] [Google Scholar]
- Doi T., Recktenwald A., Karaki Y., Kikuchi M., Morikawa K., Ikehara M., Inaoka T., Hori N., Ohtsuka E. Role of the basic amino acid cluster and Glu-23 in pyrimidine dimer glycosylase activity of T4 endonuclease V. Proc Natl Acad Sci U S A. 1992 Oct 15;89(20):9420–9424. doi: 10.1073/pnas.89.20.9420. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hori N., Doi T., Karaki Y., Kikuchi M., Ikehara M., Ohtsuka E. Participation of glutamic acid 23 of T4 endonuclease V in the beta-elimination reaction of an abasic site in a synthetic duplex DNA. Nucleic Acids Res. 1992 Sep 25;20(18):4761–4764. doi: 10.1093/nar/20.18.4761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kim J., Linn S. The mechanisms of action of E. coli endonuclease III and T4 UV endonuclease (endonuclease V) at AP sites. Nucleic Acids Res. 1988 Feb 11;16(3):1135–1141. doi: 10.1093/nar/16.3.1135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Latham K. A., Taylor J. S., Lloyd R. S. T4 endonuclease V protects the DNA strand opposite a thymine dimer from cleavage by the footprinting reagents DNase I and 1,10-phenanthroline-copper. J Biol Chem. 1995 Feb 24;270(8):3765–3771. doi: 10.1074/jbc.270.8.3765. [DOI] [PubMed] [Google Scholar]
- Manuel R. C., Latham K. A., Dodson M. L., Lloyd R. S. Involvement of glutamic acid 23 in the catalytic mechanism of T4 endonuclease V. J Biol Chem. 1995 Feb 10;270(6):2652–2661. doi: 10.1074/jbc.270.6.2652. [DOI] [PubMed] [Google Scholar]
- Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [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]
- Prince M. A., Friedman B., Gruskin E. A., Schrock R. D., 3rd, Lloyd R. S. Selective metal binding to Cys-78 within endonuclease V causes an inhibition of catalytic activities without altering nontarget and target DNA binding. J Biol Chem. 1991 Jun 5;266(16):10686–10693. [PubMed] [Google Scholar]
- Schrock R. D., 3rd, Lloyd R. S. Reductive methylation of the amino terminus of endonuclease V eradicates catalytic activities. Evidence for an essential role of the amino terminus in the chemical mechanisms of catalysis. J Biol Chem. 1991 Sep 15;266(26):17631–17639. [PubMed] [Google Scholar]
- Schrock R. D., 3rd, Lloyd R. S. Site-directed mutagenesis of the NH2 terminus of T4 endonuclease V. The position of the alpha NH2 moiety affects catalytic activity. J Biol Chem. 1993 Jan 15;268(2):880–886. [PubMed] [Google Scholar]
- Takeshita M., Chang C. N., Johnson F., Will S., Grollman A. P. Oligodeoxynucleotides containing synthetic abasic sites. Model substrates for DNA polymerases and apurinic/apyrimidinic endonucleases. J Biol Chem. 1987 Jul 25;262(21):10171–10179. [PubMed] [Google Scholar]
- Tchou J., Michaels M. L., Miller J. H., Grollman A. P. Function of the zinc finger in Escherichia coli Fpg protein. J Biol Chem. 1993 Dec 15;268(35):26738–26744. [PubMed] [Google Scholar]