A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum ΔH Journal Article
| Authors: | Kelman, Z.; Hurwitz, J. |
| Article Title: | A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum ΔH |
| Abstract: | Replication factor C (RFC, also called activator 1), in conjunction with proliferating cell nuclear antigen (PCNA), is responsible for processive DNA synthesis catalyzed by the eukaryotic replicative DNA polymerases δ and ε. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum ΔH. In contrast to the five subunit RFC complex isolated from eukaryotic cells, the mthRFC contains only two subunits. The two genes encoding the RFC subunits called, mthRFC1 and mthRFC3, were cloned, and the proteins (54.4 and 36.8 kDa, respectively) were overexpressed in Escherichia coli and purified individually and as a complex. The gene encoding PCNA was also cloned, and the protein was purified after overexpression in E. coli. Based on sizing column elution and subunit composition, the mthRFC complex appears to be a hexamer consisting of two mthRFC1 protomers and four mthRFC3 protomers. Although mthRFC differs in organization from its eukaryotic counterpart, it was shown to be functionally similar to eukaryotic RFC in: (i) catalyzing DNA-dependent ATP hydrolysis; (ii) binding preferentially to DNA primer ends; (iii) loading mthPCNA onto singly nicked circular DNA; and (iv) supporting mthPolB-catalyzed PCNA-dependent DNA chain elongation. The importance and roles of RFC and PCNA in M. thermoautotrophicum ΔH replication are discussed. |
| Keywords: | sequence analysis; dna-binding proteins; nonhuman; dna polymerase; dna replication; homeodomain proteins; bacteria (microorganisms); dna; amino acid sequence; molecular sequence data; eukaryota; escherichia coli; recombinant proteins; saccharomyces cerevisiae proteins; replication factor c; cycline; protein subunit; replication protein c; eukaryotic cell; protein structure; structure analysis; repressor proteins; adenosine triphosphatases; proto-oncogene proteins c-bcl-2; archaea; proliferating cell nuclear antigen; dna-directed dna polymerase; methanobacterium thermoautotrophicum; methanobacterium; circular dna; priority journal; article; methanothermobacter thermautotrophicus |
| Journal Title: | Journal of Biological Chemistry |
| Volume: | 275 |
| Issue: | 10 |
| ISSN: | 0021-9258 |
| Publisher: | American Society for Biochemistry and Molecular Biology |
| Date Published: | 2000-03-10 |
| Start Page: | 7327 |
| End Page: | 7336 |
| Language: | English |
| DOI: | 10.1074/jbc.275.10.7327 |
| PUBMED: | 10702304 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Export Date: 18 November 2015 -- Source: Scopus |
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