Abstract
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.
Highlights
The typical archaeal MCM exhibits helicase activity independently in vitro
The assembly of pre-replication complexes (pre-RCs) begins with the association of the ORC (Origin recognition complex) with DNA, followed by the sequential recruitment of Cdc[6], Cdt[1] and MCM2-7, with ORCCdc6-mediated ATP hydrolysis facilitating the loading of the MCM heterohexamer
The sequence of the protein was analyzed using the Conserved Domains Database (CDD; www.ncbi. nlm.nih.gov/cdd), and the analysis predicted several domains typically found in archaeal MCM proteins, including the MCM2/3/ 5 superfamily domain
Summary
The typical archaeal MCM exhibits helicase activity independently in vitro. This study characterizes MCM from the euryarchaeon Picrophilus torridus. 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. No helicase loader has been clearly identified in archaea, though in vitro evidence suggests that the Orc1/Cdc[6] protein may serve as both initiator and helicase loader[3,4]. The assembly of pre-RCs begins with the association of the ORC (Origin recognition complex) with DNA, followed by the sequential recruitment of Cdc[6], Cdt[1] and MCM2-7, with ORCCdc6-mediated ATP hydrolysis facilitating the loading of the MCM heterohexamer. We find that at pH 4 GINS stimulates the ability of MCM to hydrolyze ATP, most dramatically at near physiological concentrations of ATP
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