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 turno ver rates were measured in a variety of dideoxynucleoside monophosphat es containing free radical-induced base modifications. The modified ba ses 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 ei ther a thymine or a cytosine base. The turnover rate for dinucleoside monophosphates containing 4,8-dihydro-4-hydroxy-8-oxo-guanine modifica tions, which are induced by singlet oxygen, were also measured. A mode l was devised for the hydrolysis of DNA by nuclease P1 which uses the observed turnover rates as parameters. The model predicts the abundanc e of monomers and dimers as hydrolysis proceeds. Whereas the level of monomers increases monotonically, the level of each dimer first increa ses 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 P-32-postlabeling ap plied to the measurement of DNA damage in nuclease P1 partial hydrolys ates of DNA.