Studies of the domain structure of mammalian DNA polymerase beta. Identification of a discrete template binding domain.
Characterization of the domain structure of DNA polymerase beta is reported. Large scale overproduction of the rat protein in Escherichia coli was achieved, and the purified recombinant protein was verified by sequencing tryptic peptides. This protein is both a single-stranded DNA binding protein and a DNA polymerase consisting of one polypeptide chain of 334 amino acids. As revealed by controlled proteolysis experiments, the protein is organized in two relatively protease-resistant segments linked by a short protease-sensitive region. One of these protease-resistant segments represents the NH2-terminal 20% of the protein. This NH2-terminal domain (of about 75 residues) has strong affinity for single-stranded nucleic acids. The other protease-resistant segment, representing the COOH-terminal domain of approximately 250 residues, does not bind to nucleic acids. Neither domain, tested as purified proteins, has substantial DNA polymerase activity. The results suggest that the NH2-terminal domain is principally responsible for the template binding activity of the intact protein.
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
A
A
Kumar
S G
SG
Widen
K R
KR
Williams
P
P
Kedar
R L
RL
Karpel
S H
SH
Wilson
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003488:Cyanogen Bromide; D004247:DNA / Q000378:metabolism; D004256:DNA Polymerase I / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000302:isolation & purification; D010447:Peptide Hydrolases; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D013698:Templates, Genetic
D010446:Peptide Fragments; D011994:Recombinant Proteins; D004247:DNA; D004256:DNA Polymerase I; D010447:Peptide Hydrolases; D003488:Cyanogen Bromide