Interplay between primase and replication factor C in the hyperthermophilic archaeon<i>Sulfolobus solfataricus</i>

Wu, Kangyun; Lai, Xiaoqin; Guo, Xin; Hu, Jinchuan; Xiang, Xiaoyu; Huang, Li

doi:10.1111/j.1365-2958.2006.05535.x

Cited by 13 publications

(15 citation statements)

References 35 publications

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“…Also, another two probes, overlapping O 1 and O 2 , were designed: O 1 9, 59-TATTGTAACTGCTATTGTTGACATACCAGGC-GCTTTTCTTTTGAAGACTTAATTAAA; and O 2 9, 59-CGAATAT-AATAAAAAAGATGTTAATTAAAATAATAAATAAACCTTTCACA-TAAAACAAA (overlapping regions underlined). These single-strand DNA probes were synthesized, denatured, labelled with [c-32 P]ATP by T4 polynucleotide kinase (NEB), and mixed with different concentrations of lysate of galactose-cultured T. tengcongensis, or recombinant TTE1926 or TTE1925 protein (as the negative control) for EMSA (Wu et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 6C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

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Section: Methodsmentioning

confidence: 99%

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

et al. 2009

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“…The recombinant proteins were purified on a HiTrap S column (5 ml; GE) preequilibrated with buffer B, and the column was washed with buffer C (20 mM Tris-Cl [pH 6.8], 1 M KCl, 1 mM EDTA, 1 mM DTT, and 10% [wt/vol] glycerol). S. solfataricus chromatin and replication proteins RFC, Pri1/Pri2, Dpo4, PCNA, Topo III, PolB1, GINS, FEN1, Cren7, and Sso7d2 were overproduced in E. coli and purified as described previously (8,12,15,17,25,26,35,39,43,53).…”

Section: Purification and Identification Of Akmt From S Islandicus mentioning

confidence: 99%

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

et al. 2012

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Protein lysine methylation occurs extensively in the Crenarchaeota, a major kingdom in the Archaea. However, the enzymes responsible for this type of posttranslational modification have not been found. Here we report the identification and characterization of the first crenarchaeal protein lysine methyltransferase, designated aKMT, from the hyperthermophilic crenarchaeon Sulfolobus islandicus. The enzyme was capable of transferring methyl groups to selected lysine residues in a substrate protein using S-adenosyl-L-methionine (SAM) as the methyl donor. aKMT, a non-SET domain protein, is highly conserved among crenarchaea, and distantly related homologs also exist in Bacteria and Eukarya. aKMT was active over a wide range of temperatures, from ϳ25 to 90°C, with an optimal temperature at ϳ60 to 70°C. Amino acid residues Y9 and T12 at the N terminus appear to be the key residues in the putative active site of aKMT, as indicated by sequence conservation and site-directed mutagenesis. Although aKMT was identified based on its methylating activity on Cren7, the crenarchaeal chromatin protein, it exhibited broad substrate specificity and was capable of methylating a number of recombinant Sulfolobus proteins overproduced in Escherichia coli. The finding of aKMT will help elucidate mechanisms underlining extensive protein lysine methylation and the functional significance of posttranslational protein methylation in crenarchaea.

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“…Human RFC stimulates the nuclease activity of Flap endonuclease 1 in an ATP-independent manner (26) and inhibits DNA joining by DNA ligase 1, whose phosphorylation regulates the interaction between the two proteins (27)(28)(29). S. solfataricus RFC interacts with eukaryotic-type primase (PriSL), resulting in modulation of the activities of both proteins (10). In this report, we show that RFC interacted with PolB1 with an estimated K D of 0.4 M. This value is lower than the intracellular RFC concentration, which is about 2.4 M based on an estimate of ϳ1,500 molecules of RFC in a cell (see Fig.…”

Section: Discussionmentioning

confidence: 99%

“…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.…”

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confidence: 99%

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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Interplay between primase and replication factor C in the hyperthermophilic archaeonSulfolobus solfataricus

Cited by 13 publications

References 35 publications

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis

Identification and Characterization of a Highly Conserved Crenarchaeal Protein Lysine Methyltransferase with Broad Substrate Specificity

Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

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