title: Characterization of the replication initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus.
abstract: Eukaryotic DNA replication is preceded by the assembly of prereplication complexes (pre-RCs) at or very near origins in G1 phase, which licenses origin firing in S phase. The archaeal DNA replication machinery broadly resembles the eukaryal apparatus, though simpler in form. The eukaryotic replication initiator origin recognition complex (ORC), which serially recruits Cdc6 and other pre-RC proteins, comprises six components, Orc1-6. In archaea, a single gene encodes a protein similar to both the eukaryotic Cdc6 and the Orc1 subunit of the eukaryotic ORC, with most archaea possessing one to three Orc1/Cdc6 orthologs. Genome sequence analysis of the extreme acidophile Picrophilus torridus revealed a single Orc1/Cdc6 (PtOrc1/Cdc6). Biochemical analyses show MBP-tagged PtOrc1/Cdc6 to preferentially bind ORB (origin recognition box) sequences. The protein hydrolyzes ATP in a DNA-independent manner, though DNA inhibits MBP-PtOrc1/Cdc6-mediated ATP hydrolysis. PtOrc1/Cdc6 exists in stable complex with PCNA in Picrophilus extracts, and MBP-PtOrc1/Cdc6 interacts directly with PCNA through a PIP box near its C terminus. Furthermore, PCNA stimulates MBP-PtOrc1/Cdc6-mediated ATP hydrolysis in a DNA-dependent manner. This is the first study reporting a direct interaction between Orc1/Cdc6 and PCNA in archaea. The bacterial initiator DnaA is converted from an active to an inactive form by ATP hydrolysis, a process greatly facilitated by the bacterial ortholog of PCNA, the β subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNA-interacting protein motifs in several archaeal PCNAs suggest the possibility of a similar mechanism of regulation existing in archaea. This mechanism may involve other yet to be identified archaeal proteins.
authors.affiliation: Department of Microbiology, University of Delhi South Campus, New Delhi, India.
authors.forename: Jasmine
authors.initials: J
authors.lastname: Arora
authors.forename: Kasturi
authors.initials: K
authors.lastname: Goswami
authors.forename: Swati
authors.initials: S
authors.lastname: Saha
mesh_terms: D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D019295:Computational Biology; D004261:DNA Replication; D019641:DNA, Archaeal / Q000378:metabolism; D051738:Origin Recognition Complex / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism*; D011485:Protein Binding; D055503:Protein Multimerization; D019603:Thermoplasmales / Q000235:genetics* / Q000378:metabolism*
chemical_list: D019843:Archaeal Proteins; D019641:DNA, Archaeal; D051738:Origin Recognition Complex; D018809:Proliferating Cell Nuclear Antigen