Characterization of the replication initiator Orc1/Cdc6 from the Archaeon Picrophilus torridus.

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 ß 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.

Key residues in Mycobacterium tuberculosis protein kinase G play a role in regulating kinase activity and survival in the host.

Protein kinase G (PknG) in Mycobacterium tuberculosis has been shown to modulate phagosome-lysosome fusion. The protein has three distinct domains, an N-terminal Trx domain, a kinase domain, and a C-terminal TPR domain. The present study extensively analyzes the roles of these domains in regulating PknG kinase activity and function. We find that the kinase domain of PknG by itself is inactive, signifying the importance of the flanking domains. Although the deletion of the Trx domain severely impacts the activity of the protein, the C-terminal region also contributes significantly in regulating the activity of the kinase. Apart from this, PknG kinase activity is dependent on the presence of threonine 309 in the p + 1 loop of the activation segment. Mutating the conserved cysteine residues in the Trx motifs makes PknG refractory to changes in the redox environment. In vitro experiments identify threonine 63 as the major phosphorylation site of the protein. Importantly, we find that this is the only site in the protein that is phosphorylated in vivo. Macrophage infection studies reveal that the first 73 residues, the Trx motifs, and the threonine 63 residue are independently essential for modulating PknG-mediated survival of mycobacteria in its host. We have extended these studies to investigate the role of PknG and PknG mutants in the pathogenesis of mycobacteria in mice. Our results reinforce the findings from the macrophage infection experiments, and for the first time demonstrate that the expression of PknG in non-pathogenic mycobacteria allows the continued existence of these bacteria in host tissues.

Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus.

The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. While PtMCM hydrolyzes ATP in DNA-independent manner, it displays very poor ability to unwind DNA independently, and then too only under acidic conditions. The protein exists stably in complex with PtGINS in whole cell lysates, interacting directly with PtGINS under neutral and acidic conditions. GINS strongly activates MCM helicase activity, but only at low pH. In consonance with this, PtGINS activates PtMCM-mediated ATP hydrolysis only at low pH, with the amount of ATP hydrolyzed during the helicase reaction increasing more than fifty-fold in the presence of GINS. While the stimulation of MCM-mediated helicase activity by GINS has been reported in MCMs from P.furiosus, T.kodakarensis, and very recently, T.acidophilum, to the best of our knowledge, this is the first report of an MCM helicase demonstrating DNA unwinding activity only at such acidic pH, across all archaea and eukaryotes. PtGINS may induce/stabilize a conducive conformation of PtMCM under acidic conditions, favouring PtMCM-mediated DNA unwinding coupled to ATP hydrolysis. Our findings underscore the existence of divergent modes of replication regulation among archaea and the importance of investigating replication events in more archaeal organisms.