Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.
FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.
Doronin
S V
SV
Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of the Russian Academy of Sciences.
Dobrikov
M I
MI
Lavrik
O I
OI
D000345:Affinity Labels; D001483:Base Sequence; D002384:Catalysis; D003432:Cross-Linking Reagents; D004257:DNA Polymerase II / Q000737:chemistry* / Q000378:metabolism; D019915:DNA Primase; D004277:DNA, Single-Stranded / Q000378:metabolism; D003845:Deoxycytosine Nucleotides / Q000737:chemistry*; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000378:metabolism; D012316:RNA Nucleotidyltransferases / Q000737:chemistry*
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004277:DNA, Single-Stranded; D003845:Deoxycytosine Nucleotides; D009097:Multienzyme Complexes; C078023:exo-N-(2-(4-azidotetrafluorobenzamido)ethyl)-deoxycytidine-5'-triphosphate; D019915:DNA Primase; D012316:RNA Nucleotidyltransferases; D004257:DNA Polymerase II