Archaeal eukaryote-like Orc1/Cdc6 initiators physically interact with DNA polymerase B1 and regulate its functions

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.

DarR, a TetR-like Transcriptional Factor, Is a Cyclic Di-AMP-responsive Repressor in Mycobacterium smegmatis

“…SPR analysis was conducted on a Biacore 3000 instrument according to previously published procedures (21). Purified c-di-AMP, c-di-GMP, or ATP diluted in reaction buffer (50 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl 2 ) to concentrations of 10, 20, or 50 M was used as the ligand and passed over the chips.…”

Radiation-sensitive Gene A (RadA) Targets DisA, DNA Integrity Scanning Protein A, to Negatively Affect Cyclic Di-AMP Synthesis Activity in Mycobacterium smegmatis

“…Surface Plasmon Resonance Analysis-Interaction between MsDisA and MsRadA in vitro was analyzed on a BIAcore 3000 instrument (GE Healthcare) as described previously (18,19). Briefly, the purified GST-tagged MsRadA fusion protein, to be used as the ligand, was diluted in HBS buffer (10 mM Hepes (pH 7.4), 150 mM NaCl, 50 M EDTA, 0.005% BIAcore surfactant P20).…”

Archaeal Eukaryote-Like Serine/Threonine Protein Kinase Interacts with and Phosphorylates a Forkhead-Associated-Domain-Containing Protein

“…Prokaryotic recombinant vectors expressing the genes for archaeal proteins and mutant proteins were constructed. E. coli BL21 CodonPlus(DE3)-RIL (Novagen) was used as the host strain to express archaeal proteins as described previously (34). Protein concentrations were determined by spectrophotometric absorbance at 260 nm, as described by Gill and Hippel (7).…”