CELL STRUCTURE AND FUNCTION 9, 83-90 (1984) C by Japan Society for Cell Biology Tight Association of DNA Polymerase ƒ¿ with Granular Structures in the Nuclear Matrix of Chick Embryo Cell: Immunocytochemical Detection with Monoclonal Antibody against DNA Polymerase ƒ¿

Immunofluorescent methods using a monoclonal antibody against chick DNA polymerase a and a rabbit antibody against chick DNA polymerase ƒÀ demonstrated that both DNA polymerases a and ƒÀ are present mainly in nuclei of cultured chick embryo cells. Fluorescence produced by anti-DNA polymerase ƒ¿ was more intense in the small granules than in other parts of the nucleus but, fluorescence produced by anti-DNA polymerase ƒÀ was distributed evenly in the nucleus. Cells first were treated with Nonidet P-40, followed by treatment with 50ƒÊg/ml pancreatic DNase and 2 M NaCl in order to prepare the nuclear matrix. Fluorescence produced by anti-DNA polymerase ƒ¿ was still detectable in the granules after these treatments, but most of the fluorescence produced by anti-DNA polymerase ƒÀ disappeared. Our results indicate that a part of DNA polymerase ƒ¿ is tightly bound to a special structure present in the nuclear matrix which presumably is the DNA replication machinery. Eukaryotic DNA polymerase a has been confirmed to function in the DNA replication, and DNA polymerase ƒÀ in DNA repair (3, 6). Recent studies with specific inhibitors of DNA polymerases have suggested that DNA polymerase ƒ¿ also may act in DNA repair (5, 13). Because the DNA synthesis involved in replication and repair may take place in different nuclear areas, the distributions of these enzymes should differ in the subcellular structures and among cells grown under different physiological conditions. The nuclear locations of both DNA polymerase ƒ¿ andƒÀ have been estabolished by biochemical methods (4, 8, 9, 14) and by immunocytochemical methods that use monoclonal antibodies against DNA polymerase ƒ¿ (1, 11) and ƒ¿ rabbit antiserum against DNA polymerase ƒÀ (11). Furthermore, the amount of DNA polymerase ƒ¿ decreases when cultured cells reach confluence (1, 11) and when cells move from the growth to the differentiated stage (11). In contrast, the content of DNA polymerase ƒÀ remains almost constant after confluence or differentiation (11). Abbreviations used: DNA polymerase, deoxynucleosidetriphosphate: DNA deoxynucleotidyltransferase (EC 2.7.7.7); FCS, fetal calf serum; PBS(-—), phosphate buffered saline without Mg2+ and Ca2+; IgG, immunoglobulin G; FITC, fluoresceinisothiocyanate; MES, 2[N-morpholino] ethane sulfonic acid; EGTA, ethyleneglycol-bis-(ƒÀ-aminoethylether) N,N,N',N'-tetraacetic acid; SDS, sodium dodecylsulfate; NP-40, Nonidet P-40.