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Crenarchaea DNA replication initiation mechanisms

-We describe the identification of a chromosomal DNA replication origin (oriC) from the hyperthermophilic archaeon Sulfolobus solfataricus (subdomain of Crenarchaeota). A candidate oriC was mapped within a 1.12-kb region located between the two divergently transcribed MCM- and cdc6-like genes. We demonstrated that plasmids containing the Sulfolobus oriC sequence and a hygromycin-resistance selectable marker were maintained in an episomal state in transformed S. solfataricus cells under selective pressure. The proposed location of the origin was confirmed by 2-D gel electrophoresis experiments. This is the first report on the functional cloning of a chromosomal oriC from an archaeon and represents an important step toward the reconstitution of an archaeal in vitro DNA replication system.

- We report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 3 Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.

- We report that a Cdc6 (cell-division control)- like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions. A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known. This allowed us to design two truncated forms of SsoCdc6-2: the delta-C (residues 1 297, molecular mass 35 kDa) and the delta-N (residues 298 400, molecular mass 11 kDa) proteins. The delta-C protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP. On the other hand, the delta-N protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2. These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity. Moreover, it was demonstrated that a Cdc6-like factor from the hyperthermophilic crenarchaea Sulfolobus solfataricus stimulates binding of the homohexameric MCM-like complex to bubble- and fork-containing DNA oligonucleotides that mimic early replication intermediates. This function does not require the Cdc6 ATP and DNA binding activities.

- The biochemical characterization of Sso Cdc6-1 and Cdc6-3 has been done. It has been found that either Sso Cdc6-1 or Cdc6-3 behave as monomers in solutions. Both factors are able to bind to various single-stranded and double-stranded DNA ligands, but Sso Cdc6-3 shows a higher DNA-binding affinity. It has also been observed that either Sso Cdc6-1 or Cdc6-3 inhibit the DNA unwinding activity of the S. solfataricus homo-hexameric mini-chromosome maintenance (MCM)-like DNA helicase (Sso MCM); although they strongly stimulate the interaction of the Sso MCM with bubblecontaining synthetic oligonucleotides. Sso Cdc6-2 physically interacts with either Sso Cdc6-1 or Sso Cdc6-3.

- We report the identification of amino acids of the Sulfolobus solfataricus mini-chromosome maintenance (MCM)-like DNA helicase (SsoMCM), which are critical for DNA binding/remodeling. A structure-guided alignment of the M. thermoautotrophicum and S. solfataricus MCM sequences identified positively charged amino acids in SsoMCM that could point to the center of the channel. These residues (Lys-129, Lys-134, His-146, and Lys-194) were changed to alanine. The purified mutant proteins were all found to form homo-hexamers in solution and to retain full ATPase activity. K129A, H146A, and K194A SsoMCMs are unable to bind DNA either in single- or double-stranded form in band shift assays and do not display helicase activity. In contrast, the substitution of lysine 134 to alanine affects only binding to duplex DNA molecules, whereas it has no effect on binding to single-stranded DNA and on the DNA unwinding activity.

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