Selective hydrolysis of damaged DNA by nuclease P1.
The turnover rates for hydrolysis by nuclease P1 of the 16 unmodified dideoxynucleoside monophosphates were measured. In addition, the turnover rates were measured in a variety of dideoxynucleoside monophosphates containing free radical-induced base modifications. The modified bases included cis-5,6-dihydroxy-5,6-dihydrothymine (thymine glycol), 5,6-dihydrothymine, 5-hydroxymethyuracil, 8-hydroxyguanine, 5-hydroxy-5-methylhydantoin and the formamido remnant which can be derived from either a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifications, which are induced by singlet oxygen, were also measured. A model was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundance of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increases and then falls off. There are advantages to phosphorylating dimers, as compared with monomers, using polynucleotide kinase. Consequently this model may be of interest in connection with 2-P-postlabeling applied to the measurement of DNA damage in nuclease P1 partial hydrolysates of DNA.
Falcone
J M
JM
Biophysics Department, Roswell Park Cancer Institute, Buffalo, NY 14263, USA.
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D004249:DNA Damage; D005609:Free Radicals / Q000737:chemistry; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D015719:Single-Strand Specific DNA and RNA Endonucleases / Q000378:metabolism*
D005609:Free Radicals; D009838:Oligodeoxyribonucleotides; D015719:Single-Strand Specific DNA and RNA Endonucleases