Specific interactions of three proliferating cell nuclear antigens with replication‐related proteins in <i>Aeropyrum pernix</i>

Imamura, K; Fukunaga, Kenzo; Kawarabayasi, Yutaka; Ishino, Yuji

doi:10.1111/j.1365-2958.2007.05645.x

Cited by 20 publications

(26 citation statements)

References 38 publications

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“…We found that RFC stimulated the polymerase and 3=-5= exonuclease activities of the polymerase in an ATP-independent manner. Similar observations were made earlier on RFC from the crenarchaeon Aeropyrum pernix, which significantly enhanced primer extension by Pol I and Pol II, two family B DNA polymerases (30). More recently, it was reported that the rate of nucleotide incorporation by PolB1 on a primed template at 60°C in the presence of RFC was nearly twice as high as that in the absence of the clamp loader in the S. solfa- 32 P-labeled p42/t76 (2 nM) in the presence or absence of RFC.…”

Section: Discussionsupporting

confidence: 70%

Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

et al. 2014

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bReplication factor C (RFC) is known to function in loading proliferating cell nuclear antigen (PCNA) onto primed DNA, allowing PCNA to tether DNA polymerase for highly processive DNA synthesis in eukaryotic and archaeal replication. In this report, we show that an RFC complex from the hyperthermophilic archaea of the genus Sulfolobus physically interacts with DNA polymerase B1 (PolB1) and enhances both the polymerase and 3=-5= exonuclease activities of PolB1 in an ATP-independent manner. Stimulation of the PolB1 activity by RFC is independent of the ability of RFC to bind DNA but is consistent with the ability of RFC to facilitate DNA binding by PolB1 through protein-protein interaction. These results suggest that Sulfolobus RFC may play a role in recruiting DNA polymerase for efficient primer extension, in addition to clamp loading, during DNA replication.A ll forms of cellular life replicate their chromosomal DNA in a strikingly similar fashion, employing clamp loader proteins to assemble ring-shaped sliding clamps in ATP-dependent reactions at new RNA-primed sites, where the clamp tethers DNA polymerase for highly processive DNA synthesis (1). Although clamp loader proteins of different origins differ at the amino acid sequence level and in subunit composition, they share both overall structures and molecular mechanisms in clamp-loading processes (2). In Escherichia coli, the clamp loader, known as the ␥ complex, consists of five subunits, i.e., ␦, ␦=, and three copies of /␥ (3). In eukarya, the clamp loader, referred to as replication factor C (RFC), has four different small subunits (RFC S ) and one large subunit (RFC L ) (2). Archaea, the third domain of life, replicate DNA in a eukaryotic-like fashion. While most of the archaeal clamp loaders are a pentameric complex of one large subunit and four identical small subunits (4-6), RFCs from some archaea exhibit variations in subunit composition. For example, RFC from Methanosarcina acetivorans shows a stoichiometry of one large subunit to three small subunits to one even smaller subunit (7).PCNA loading by the RFC heteropentamer (1 RFC L /4 RFC S ) from the thermoacidophilic crenarchaeon Sulfolobus solfataricus has been extensively studied (8,9). Unlike eukaryotes and euryarchaea, which possess a homotrimeric PCNA, crenarchaea have an unusual heterotrimeric PCNA comprising PCNA1, PCNA2, and PCNA3 (8). The S. solfataricus RFC has been shown to interact with the PCNA1 and PCNA2 subunits through RFC S and with PCNA3 through RFC L (8). Opening of the PCNA ring by RFC at the PCNA3-PCNA1 interface allows the passage of DNA into the PCNA ring (9). In addition to its role in PCNA loading, RFC also interacts with the eukaryotic-type primase (10). As the result of this interaction, primer synthesis by the primase is inhibited while the ATPase activity of RFC is stimulated, suggesting that RFC may serve roles in regulating primer synthesis and transfer of primers to DNA polymerase.In the present study, we show that Sulfolobus RFC interacts physically with DNA polymera...

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Section: Discussionsupporting

confidence: 70%

Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

et al. 2014

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“…Determination of the content of each PCNA protein by Western blot analysis has indicated that the three PCNA proteins are expressed at different levels in the crenarchaeon A. pernix (Imamura et al, 2007). Doing the same for S. islandicus has yielded similar results (unpublished data).…”

Section: Discussionsupporting

confidence: 60%

“…While eukaryotes and euryarchaea employ a homotrimeric PCNA ring, crenarchaea encode three different PCNA subunits that may form trimeric rings of different compositions. It has been shown that the heterotrimeric form is the only form that supports DNA replication in vitro (Dionne et al, 2003), although other trimeric forms have also been observed in vitro for Aeropyrum pernix and Sulfolobus tokodai (Imamura et al, 2007;Lu et al, 2008). This raises an important question as to whether there is functional redundancy or differentiation of crenarchaeal PCNAs.…”

Section: Introductionmentioning

confidence: 99%

Revealing the essentiality of multiple archaeal pcna genes using a mutant propagation assay based on an improved knockout method

et al. 2010

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Organisms belonging to the Crenarchaeota lineage contain three proliferating cell nuclear antigen (PCNA) subunits, while those in the Euryarchaeota have only one, as for Eukarya. To study the mechanism of archaeal sliding clamps, we sought to generate knockouts for each pcna gene in Sulfolobus islandicus, a hyperthermophilic crenarchaeon, but failed with two conventional knockout methods. Then, a new knockout scheme, known as marker insertion and target gene deletion (MID), was developed, with which transformants were obtained for each pMID-pcna plasmid. We found that mutant cells persisted in transformant cultures during incubation of pMIDpcna3 and pMID-araS-pcna1 transformants under counter selection. Studying the propagation of mutant cells by semiquantitative PCR analysis of the deleted target gene allele (Dpcna1 or Dpcna3) revealed that mutant cells could no longer be propagated, demonstrating that these pcna genes are absolutely required for host cell viability. Because the only prerequisite for this assay is the generation of a MID transformant, this approach can be applied generally to any micro-organisms proficient in homologous recombination.

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“…However, a PCNA-forming heterotrimer (PCNA123), consisting of PCNA1, PCNA2 and PCNA3, has been found in three crenarchaeal species: Sulfolobus solfataricus (ssoPCNA) (Dionne et al, 2003), Aeropyrum pernix (apePCNA) (Imamura et al, 2007), and Sulfolobus tokodaii (stoPCNA) (Lu et al, 2008). The PCNA123 heterotrimer plays a similar role to eukaryotic PCNA in that it affects the activities of DNA polymerase (PolB1), DNA Ligase I, and flap endonuclease I (Fen-I) (Dionne et al, 2003).…”

Section: Introductionmentioning

confidence: 99%