Biochemistry of terminal deoxynucleotidyltransferase. Identification and unity of ribo- and deoxyribonucleoside triphosphate binding site in terminal deoxynucleotidyltransferase.
Terminal deoxynucleotidyltransferase is the only DNA polymerase that is strongly inhibited in the presence of ATP. We have labeled calf terminal deoxynucleotidyltransferase with [32P]ATP in order to identify its binding site in terminal deoxynucleotidyltransferase. The specificity of ATP cross-linking to terminal deoxynucleotidyltransferase is shown by the competitive inhibition of the overall cross-linking reaction by deoxynucleoside triphosphates, as well as the ATP analogs Ap4A and Ap5A. Tryptic peptide mapping of [32P]ATP-labeled enzyme revealed a peptide fraction that contained the majority of cross-linked ATP. The properties, chromatographic characteristics, amino acid composition, and sequence analysis of this peptide fraction were identical with those found associated with dTTP cross-linked terminal deoxynucleotidyl-transferase peptide (Pandey, V. N., and Modak, M. J. (1988a). J. Biol. Chem. 263, 3744-3751). The involvement of the same 2 cysteine residues in the crosslinking of both nucleotides further confirmed the unity of the ATP and dTTP binding domain that contains residues 224-237 in the primary amino acid sequence of calf terminal deoxynucleotidyltransferase.
Pandey
V N
VN
Department of Biochemistry, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark 07103.
Modak
M J
MJ
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D001665:Binding Sites; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism* / Q000528:radiation effects; D003854:Deoxyribonucleotides / Q000494:pharmacology*; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D012265:Ribonucleotides / Q000494:pharmacology*; D014466:Ultraviolet Rays
D000596:Amino Acids; D003854:Deoxyribonucleotides; D010446:Peptide Fragments; D012265:Ribonucleotides; D000255:Adenosine Triphosphate; D004253:DNA Nucleotidylexotransferase