A unique organization of the protein subunits of the DNA polymerase clamp loader in the archaeon Methanobacterium thermoautotrophicum deltaH.
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 delta and epsilon. Here we report the isolation and characterization of homologues of RFC and PCNA from the archaeon, Methanobacterium thermoautotrophicum DeltaH. 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 DeltaH replication are discussed.
Kelman
Z
Z
Department of Molecular Biology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. z-kelman@ski.mskcc.org
Hurwitz
J
J
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000595:Amino Acid Sequence; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D018398:Homeodomain Proteins; D017014:Methanobacterium / Q000235:genetics*; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics* / Q000302:isolation & purification; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins / Q000302:isolation & purification; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D018809:Proliferating Cell Nuclear Antigen; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D000251:Adenosine Triphosphatases; D051818:Replication Protein C