Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells.
The elongation of primed DNA templates by DNA polymerase delta and DNA polymerase epsilon requires the action of two accessory proteins, proliferating cell nuclear antigen and activator 1 (A1, also called replication factor C). A1 is an enzyme that contains five different subunits (145, 40, 38, 37, and 36.5 kDa). In this paper, we describe the isolation of the gene encoding the 37-kDa subunit from HeLa cells. This gene was cloned, sequenced, and overexpressed in Escherichia coli. The amino acid sequence shows a high degree of homology to the 40-kDa subunit of A1; they both contain the identical ATP-binding motif, but in contrast to the bacterial expressed 40-kDa protein, the 37-kDa expressed protein did not bind ATP. Both the 37- and 40-kDa proteins share substantial homology with the phage T4 gene 44 protein and to a lesser extent with the tau and gamma subunits of the E. coli DNA polymerase III holoenzyme. Polyclonal antibodies against the bacterially expressed 37- and 40-kDa proteins do not crossreact and are specific in their interaction. Antibodies against the 37-kDa protein maximally inhibited (by 50%) the A1-dependent synthesis of DNA by DNA polymerase delta; antibodies against the 40-kDa protein quantitatively inhibited the same reaction. When A1-dependent synthesis of DNA was partially inhibited by antibodies against the 40-kDa subunit, the addition of antibodies against the 37-kDa subunit inhibited DNA synthesis to a greater extent than the anti-37-kDa antibody alone. These results suggest that both the 37- and 40-kDa subunits of A1 are required for the biological role of A1 and that they may function differently in this process.
Graduate Program in Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.
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Hurwitz
D000595:Amino Acid Sequence; D000906:Antibodies; D001483:Base Sequence; D003001:Cloning, Molecular; D004261:DNA Replication / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009500:Neutralization Tests; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D011401:Promoter Regions, Genetic; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D013698:Templates, Genetic
D000906:Antibodies; C096816:BCL2-related protein A1; D004273:DNA, Neoplasm; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D046911:Macromolecular Substances; D015778:Minor Histocompatibility Antigens; D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; C497563:RFC4 protein, human; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D051818:Replication Protein C