Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells Journal Article
| Authors: | Chen, M.; Pan, Z. Q.; Hurwitz, J. |
| Article Title: | Studies of the cloned 37-kDa subunit of activator 1 (replication factor C) of HeLa cells |
| Abstract: | The elongation of primed DNA templates by DNA polymerase δ and DNA polymerase F 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 τ and γ 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 δ 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. |
| Keywords: | gene sequence; human cell; dna-binding proteins; comparative study; dna polymerase; dna replication; gene expression; gene library; hela cell; hela cells; bacteria (microorganisms); nuclear proteins; molecular cloning; cloning, molecular; amino acid sequence; molecular sequence data; screening; escherichia coli; dna, neoplasm; immunoblotting; simian virus 40; base sequence; antibodies; sequence homology, nucleic acid; enzyme subunit; molecular weight; dna synthesis inhibition; protein antibody; oligodeoxyribonucleotides; proliferating cell nuclear antigen; virus dna; dna template; templates, genetic; promoter regions (genetics); cell nucleus antigen; neutralization tests; simiae; simian virus; human; priority journal; article; support, non-u.s. gov't; support, u.s. gov't, p.h.s.; macromolecular systems; dna polymerases δ and ε; simian virus 40 dna replication |
| Journal Title: | Proceedings of the National Academy of Sciences of the United States of America |
| Volume: | 89 |
| Issue: | 12 |
| ISSN: | 0027-8424 |
| Publisher: | National Academy of Sciences |
| Date Published: | 1992-06-15 |
| Start Page: | 5211 |
| End Page: | 5215 |
| Language: | English |
| DOI: | 10.1073/pnas.89.12.5211 |
| PUBMED: | 1351677 |
| PROVIDER: | scopus |
| PMCID: | PMC49261 |
| DOI/URL: | |
| Notes: | Source: Scopus |
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