Identification and subcellular localization of the polypeptide for chick DNA primase with a specific monoclonal antibody.

Abstract:

Polypeptides responsible for activities of chick embryo DNA primase and DNA polymerase alpha were identified using monoclonal antibodies specific to these two enzymes. The 4-8H antibody neutralized DNA polymerase alpha activity measured on activated DNA template and also ribonucleoside triphosphate-dependent DNA synthesis on single-stranded DNA template (DNA primase-DNA polymerase alpha combined activity) to a partial extent (about 30%), but did not affect DNA primase activity. The 4-2D antibody, although it did not affect DNA polymerase alpha activity, did neutralize both DNA primase activity and DNA primase-DNA polymerase alpha combined activity extensively (up to 70%). Immunoblotting analysis of the DNA primase-DNA polymerase alpha complex showed that 4-2D and 4-8H antibodies recognize 60-kDa and 160-180-kDa polypeptides, respectively. An immunoaffinity column made of either of these antibodies retained DNA primase-DNA polymerase alpha complex. When the enzyme was eluted from the 4-8H column with alkaline solution, DNA primase was eluted prior to DNA polymerase alpha. In the case of 4-2D antibody column chromatography, the elution order of two enzymes was reversed. Results indicate that two enzymes in the complex which was retained in the antibody column were dissociated by lower alkaline pH than that dissociated the antigenic enzymes from the corresponding antibodies. In both cases, the fractions with DNA primase activity contained exclusively 60-kDa polypeptide, while those with DNA polymerase alpha contained 160-180-kDa polypeptides. Thus, DNA primase resided in 60-kDa polypeptide and was recognized by 4-2D antibody while DNA polymerase alpha resided in 160-180-kDa polypeptides and was recognized by 4-8H antibody. Immunofluorescence made with the DNA primase-specific 4-2D antibody as well as with 4-8H antibody appeared in granular structures which were tightly bound to the nuclear matrix. These nuclear fluorescences were much reduced in quiescent cells. Furthermore, since the fluorescence made by these antibodies was induced by adding serum to the quiescent cells in serum-deprived cultures, the expression of DNA primase and its organization in the structures on the nuclear matrix are regulated in correlation to the proliferating stage of cells, as observed with DNA polymerase alpha.

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