Purification and substrate specificity of polydeoxyribonucleotide kinases isolated from calf thymus and rat liver.
Damage to DNA can result in strand breaks with 5'-hydroxyl and 3'-phosphate termini. Before DNA polymerases and ligases can rejoin the broken strands, such termini have to be restored to 5'-phosphate and 3'-hydroxyl groups. Polydeoxynucleotide kinase is an enzyme that may fulfil this function. We have purified the kinases from calf thymus and rat liver to near homogeneity. Based on SDS-polyacrylamide gel electrophoresis and activity gels, the enzymes from both sources are approximately 60-kDa polypeptides. Both enzymes have an acidic pH optimum (5.5-6.0) for kinase activity, and similar pl values (8.5-8.6), and a specificity for DNA. The calf thymus kinase possesses a 3'-phosphatase activity, as has previously been shown for the rat liver enzyme. The minimum size of oligonucleotide that can be labelled is 7-8 nucleotides in length, but the optimal size appears to be > 18 nucleotides. Comparison of phosphorylation of oligo(dA)24 and oligo(dT)24 with oligonucleotides containing a varied nucleotide sequence indicated that the homopolymers are poorer substrates. Unlike the bacteriophage T4 polynucleotide kinase, the mammalian kinases exhibit no preference for 5'-overhanging termini when acting at DNA termini produced by restriction enzymes. With double-stranded oligonucleotide complexes designed to mode single-strand gaps and nicks, the mammalian kinases preferentially phosphorylate the 5'-terminus associated with the gap or nick, in keeping with the idea that the kinases are involved in the repair of DNA single-strand breaks.
Karimi-Busheri
F
F
Experimental Oncology, Cross Cancer Institute, Edmonton, Alberta, Canada.
Weinfeld
M
M
D000818:Animals; D002417:Cattle; D002457:Cell Extracts; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008099:Liver / Q000201:enzymology*; D010766:Phosphorylation; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D013379:Substrate Specificity; D013950:Thymus Gland / Q000201:enzymology*
D002457:Cell Extracts; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase