Identification and properties of the catalytic domain of mammalian DNA polymerase beta.
Rat DNA polymerase beta (beta-pol) is a 39-kDa protein organized in two tightly folded domains, 8-kDa N-terminal and 31-kDa C-terminal domains, connected by a short protease-sensitive region. The 8-kDa domain contributes template binding to the intact protein, and we now report that the 31-kDa C-terminal domain contributes catalytic activity. Our results show that this domain as a purified proteolytic fragment conducts DNA synthesis under appropriate conditions but the kcat is lower and primer extension properties are different from those of the intact enzyme. A proteolytic truncation of the 31-kDa catalytic domain fragment, to remove a 60-residue segment from the NH2-terminal end, results in nearly complete loss of activity, suggesting the importance of this segment. Overall, these results indicate that the domains of beta-pol have distinct functional roles, template binding and nucleotidyltransferase, respectively; yet, the intact protein is more active for each function than the isolated individual domain fragment.
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
A
A
Kumar
J
J
Abbotts
E M
EM
Karawya
S H
SH
Wilson
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D004256:DNA Polymerase I / Q000737:chemistry* / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000378:metabolism; D015394:Molecular Structure; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D004256:DNA Polymerase I