REPAIR OF DNA STRAND GAPS AND NICKS CONTAINING 3'-PHOSPHATE AND 5'-HYDROXYL TERMINI BY PURIFIED MAMMALIAN ENZYMES

A putative role for mammalian polynucleotide kinases that possess both 5'-phosphotransferase and 3'-phosphatase activity is the restoration of DNA strand breaks with 5'-hydroxyl termini or 3'-phosphate termini, or both, to a form that supports the subsequent action of DNA repair polymerases and DNA ligases, i.e. 5'-phosphate and 3'-hydroxyl termini , To further assess this possibility, we compared the activity of the 3'-phosphatase of purified calf thymus polynucleotide kinase towards a variety of substrates, The rate of removal of 3'-phosphate groups fro m nicked or short (1 nt) gapped sites in double-stranded DNA was obser ved to be similar to that of 3'-phosphate groups from single-stranded substrates, Thus this activity of polynucleotide kinase does not appea r to be influenced by steric accessibility of the phosphate group. We subsequently demonstrated that the concerted reactions of polynucleoti de kinase and purified human DNA ligase I could efficiently repair DNA nicks possessing 3'-phosphate and 5'-hydroxyl termini, and similarly the combination of these two enzymes together with purified rat DNA po lymerase beta could seal a strand break with a 1 nt gap, With a substr ate containing a nick bounded by 3'- and 5'-OH termini, the rate of ga p filling by polymerase beta was significantly enhanced in the presenc e of polynucleotide kinase and ATP, indicating the positive influence of 5'-phosphorylation. The reaction was further enhanced by addition o f DNA ligase I to the reaction mixture. This is due, at least in part, to an enhancement by DNA ligase I of the rate of 5'-phosphorylation c atalyzed by polynucleotide kinase.