Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain.
DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.
Recupero
A J
AJ
Department of Biological Sciences, University of Cincinnati, Ohio 45221-0006.
Rein
D C
DC
Meyer
R R
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D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D001665:Binding Sites; D015153:Blotting, Western; D004256:DNA Polymerase I / Q000737:chemistry* / Q000276:immunology; D004591:Electrophoresis, Polyacrylamide Gel; D000939:Epitopes / Q000737:chemistry; D004926:Escherichia coli / Q000201:enzymology; D005260:Female; D007075:Immunoglobulin M / Q000276:immunology; D007163:Immunosorbent Techniques; D008114:Liver Neoplasms, Experimental / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D009711:Nucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007075:Immunoglobulin M; D009711:Nucleotides; D010446:Peptide Fragments; D004256:DNA Polymerase I