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 G 1 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 ␤ subunit of Pol III. The stimulation of PtOrc1/Cdc6-mediated ATP hydrolysis by PCNA and the conservation of PCNAinteracting 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.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus

“…Accordingly, MCM was immunoprecipitated from isolated P. torridus extracts with rabbit anti-MCM antibody and immunoprecipitates analyzed, as described earlier13. Western blot analysis of the immunoprecipitates that had been resolved by SDS-PAGE revealed that GINS interacted with MCM in these extracts (Fig.…”

The multiple stress responsive transcriptional regulator Rv3334 of Mycobacterium tuberculosis is an autorepressor and a positive regulator of kstR

“…Chase experiment was carried out as described by Arora et al . . Chase experiment was carried out using [γ‐ 32 P]ATP labeled DNA substrate (500 bp).…”

“…Oligos for EMSA was annealed and then labeled with [c-32 P]ATP. Chase experiment was carried out as described by Arora et al [39]. Chase experiment was carried out using [c-32 P]ATP labeled DNA substrate (500 bp).…”

Genome Replication in Thermococcus kodakarensis Independent of Cdc6 and an Origin of Replication

“…Given the established and convincingly large body of evidence that archaeal genomes have defined origins recognized and bound by initiator proteins (Matsunaga et al, 2001 , 2007 , 2010 ; Norais et al, 2007 ; Wigley, 2009 ; Kawakami and Katayama, 2010 ; Beattie and Bell, 2011 ; Scholefield et al, 2011 ; Pelve et al, 2013 ), the proposal that RDR supports rapid growth in an archaeon (Hawkins et al, 2013 ) is unique and challenging. Most archaea encode replication initiator proteins that are homologous to eukaryotic initiation factors Orc1 and Cdc6, and one or more Cdc6-encoding genes are present in almost all sequenced archaeal genomes, usually located adjacent to a known or predicted origin(s) of replication (Robinson and Bell, 2005 ; Barry and Bell, 2006 ; Dueber et al, 2011 ; Bell, 2012 ; Makarova and Koonin, 2013 ; Samson et al, 2013 ; Arora et al, 2014 ; Wu et al, 2014 ; Cossu et al, 2015 ). An increase in the number of Cdc6 proteins is often positively correlated with the number of replication origins (Samson et al, 2013 ); H. volcanii encodes fourteen Cdc6 proteins that function at three chromosomal origins and an integrated viral origin (Norais et al, 2007 ).…”