Chromatin 3'-phosphatase/5'-OH kinase cannot transfer phosphate from 3' to 5' across a strand nick in DNA.
Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP-mediated phosphorylation of the 5'-OH end.
D000818:Animals; D001080:Apurinic Acid / Q000378:metabolism; D002843:Chromatin / Q000201:enzymology*; D011088:DNA Ligases / Q000378:metabolism; D004260:DNA Repair; D008099:Liver / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity
D002843:Chromatin; D009097:Multienzyme Complexes; D001080:Apurinic Acid; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; C050392:chromatin 3'-phosphatase-5'-hydroxy kinase; D010744:Phosphoric Monoester Hydrolases; D011088:DNA Ligases