Immunochemical detection of a primase activity related subunit of DNA polymerase. cap alpha. from human and mouse cells using the monoclonal antibody

Journal Article · Tue Dec 01 00:00:00 EST 1987 · Biochemistry; (United States)

OSTI ID:5357409

Yagura, T; Kozu, T; Seno, T; Tanaka, S

A hybrid cell line (HDR-854-Er) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase ..cap alpha.. was established by immunizing mice with DNA replicase complex (DNA polymerase ..cap alpha..-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralized the primase activity as assessed either by the direct primase assay (incorporation of (..cap alpha..-/sup 32/P)AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase ..cap alpha.. activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase ..cap alpha... The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDA) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3 S DNA polymerase ..cap alpha.. which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase ..cap alpha... Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDA polypeptide is associated with DNA polymerase ..cap alpha.., and not with the primase. These results indicate that the 77-kDa polypeptide detected with the E4 antibody is not the primase but is a subunit firmly bound to DNA polymerase ..cap alpha.. catalytic polypeptide and yet influences the activity of the associated DNA primase.

• Research Organization: Saitama Cancer Center Research Institute, Japan
• OSTI ID: 5357409
• Journal Information: Biochemistry; (United States), Vol. 26:24
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
DNA POLYMERASES
CYTOCHEMISTRY
IMMUNOASSAY
AMP
ATP
ENZYME ACTIVITY
HELA CELLS
HYBRIDOMAS
MICE
MOLECULAR STRUCTURE
MONOCLONAL ANTIBODIES
PHOSPHORUS 32
TRITIUM COMPOUNDS
ANIMAL CELLS
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMISTRY
CHEMISTRY
DAYS LIVING RADIOISOTOPES
ENZYMES
ISOTOPES
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
NUCLEI
NUCLEOTIDES
NUCLEOTIDYLTRANSFERASES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORUS-GROUP TRANSFERASES
POLYMERASES
RADIOISOTOPES
RODENTS
TRANSFERASES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques