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ABSTRACT: The roles of Y-family DNA polymerases and the regulation mechanisms are not well defined in Archaea. In this study, we performed in vitro and in vivo characterization of the physical interaction between the archaeon Sulfolobus solfataricus Y-family DNA polymerase (SsoPolY) and three eukaryote-like Orc1/Cdc6 proteins (SsoCdc6-1, SsoCdc6-2, and SsoCdc6-3). The effect of SsoCdc6-2 was the strongest, and the three SsoCdc6 proteins were shown to have very different effects on the function of SsoPolY. SsoCdc6-2 inhibited both the DNA-binding activity and DNA polymerization activity of SsoPolY on the DNA substrates containing mismatched bases, while it formed a large complex with SsoPolY and stimulated DNA-binding activity on paired primer-template DNA substrates. SsoCdc6-2 and S. solfataricus PCNA (SsoPCNA) showed a cooperative effect on polymerization by SsoPolY on paired DNA templates, but SsoCdc6 reduced the stimulating effect of SsoPCNA on this polymerization on mismatched DNA substrates. Therefore, we uncovered a DNA substrate-dependent SsoCdc6/SsoPolY interaction mechanism. This is the first evidence for a physical and functional linkage between archaeal eukaryote-like Orc1/Cdc6 proteins and Y-family DNA polymerase.
Biochemical and Biophysical Research Communications 06/2010; 396(3):755-62. · 2.48 Impact Factor
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ABSTRACT: Protein phosphorylation plays an important role in cell signaling. However, in the Archaea, little is known about which proteins are phosphorylated and which kinases are involved. In this study, we identified, for the first time, a typical eukaryote-like Ser/Thr protein kinase and its protein partner, a forkhead-associated (FHA)-domain-containing protein, from the archaeon Sulfolobus tokodaii strain 7. This protein kinase, ST1565, physically interacted with the FHA-domain-containing protein, ST0829, both in vivo and in vitro. ST1565 preferred Mn(2+) as a cofactor for autophosphorylation and for substrate phosphorylation; the optimal temperature for this was 45 degrees C, and the optimal pH was 5.5 to 7.5. The critical amino acid residues of the conserved FHA and kinase domain sites were identified by performing a series of mutation assays. Thr329 was part of a major activation site in the kinase, while Thr326 was a negative regulation site. Several mutants with amino acid substitutions in the conserved FHA domain sites of ST0829 did not physically interact with ST1565. A structural model for the FHA domain demonstrated that the mutation sites were located at the edge of the protein and thus were in the domain that potentially interacts with ST1565. This report describes pioneering work on the third domain of life, the Archaea, showing that a protein kinase interacts with and phosphorylates an FHA-domain-containing protein. Our data provide critical information on the structural or functional characteristics of archaeal proteins and could help increase our understanding of fundamental signaling mechanisms in all three domains of life.
Journal of bacteriology 04/2010; 192(7):1956-64. · 3.94 Impact Factor
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ABSTRACT: Archaeal DNA replication machinery represents a core version of that found in eukaryotes. However, the proteins essential for the coordination of origin selection and the functioning of DNA polymerase have not yet been characterized in archaea, and they are still being investigated in eukaryotes. In the current study, the Orc1/Cdc6 (SsoCdc6) proteins from the crenarchaeon Sulfolobus solfataricus were found to physically interact with its DNA polymerase B1 (SsoPolB1). These SsoCdc6 proteins stimulated the DNA-binding ability of SsoPolB1 and differentially regulated both its polymerase and nuclease activities. Furthermore, the proteins also mutually regulated their interactions with SsoPolB1. In addition, SsoPolB1c467, a nuclease domain-deleted mutant of SsoPolB1 defective in DNA binding, retains the ability to physically interact with SsoCdc6 proteins. Its DNA polymerase activity could be stimulated by these proteins. We report on a linkage between the initiator protein Orc1/Cdc6 and DNA polymerase in the archaeon. Our present and previous findings indicate that archaeal Orc1/Cdc6 proteins could potentially play critical roles in the coordination of origin selection and cell-cycle control of replication.
Proceedings of the National Academy of Sciences 06/2009; 106(19):7792-7. · 9.68 Impact Factor
