Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase.
The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in DNA.
Habraken
Y
Y
Laboratoire de Biochimie, Faculté des Sciences, Université de Liège, Belgium.
Verly
W G
WG
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002413:Cations, Divalent; D002843:Chromatin / Q000201:enzymology*; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008970:Molecular Weight; D009708:Nucleotidases / Q000302:isolation & purification* / Q000378:metabolism; D009994:Osmolar Concentration; D010770:Phosphotransferases / Q000302:isolation & purification*; D011116:Polynucleotide 5'-Hydroxyl-Kinase / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats; D014357:Trypsin
D002413:Cations, Divalent; D002843:Chromatin; D000255:Adenosine Triphosphate; D004247:DNA; D010770:Phosphotransferases; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D009708:Nucleotidases; C039170:deoxynucleotide 3'-phosphatase; D014357:Trypsin