Characterization of the MCM homohexamer from the thermoacidophilic euryarchaeon Picrophilus torridus

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.

The RecJ2 protein in the thermophilic archaeon Thermoplasma acidophilum is a 3′-5′ exonuclease that associates with a DNA replication complex

“…TaGINS stimulated the exonuclease activity of TaRecJ2 only at acidic pH values ϽpH 7.0. A similar situation was recently reported for Picrophilus torridus, in which GINS stimulates the MCM helicase activity only under acidic conditions at pH 3.0 and pH 4.0 (14). The intracellular pH values of T. acidophilum and P. torridus are reportedly 5.5 and 4.6, respectively (33,34), which are consistent with the finding that GINS efficiently functions only at acidic pH values to stimulate its partners in Thermoplasmatales.…”

“…interaction with GINS in vitro has been reported in both Euryarchaeota and Crenarchaeota, although the structural basis for the interaction between archaeal MCM and GINS is unknown (10,(12)(13)(14)(15).…”

Amidst multiple binding orientations on fork DNA, Saccharolobus MCM helicase proceeds N-first for unwinding

“…GINS binds primarily to the AAA + CTD of MCM5 bringing in Cdc45 to interact with and close the interface with MCM2, aligning the motor domains into a proper configuration for activity (Costa et al, 2011). Archaea have a single MCM protein that is equally homologous to the six eukaryotic MCM2-7 proteins (Makarova et al, 2012; Goswami et al, 2015), and in contrast to eukaryotes, the archaeal MCM helicase is active on its own in vitro and does not require accessory proteins for robust DNA unwinding (Chong et al, 2000; Marinsek et al, 2006). Archaeal MCM forms a homohexameric complex but can also interact with orthologs of Cdc45 (RecJ) and the GINS (GINS23, GINS15) complex to stimulate the helicase activity further (Yoshimochi et al, 2008; Lang and Huang, 2015; Xu et al, 2016), although Cdc45 ( i.e.…”

Insights into archaeal chaperone machinery: a network-based approach

“…Therefore, we have attempted to create and analyze the protein-protein interaction network of P. torridus chaperones in the current study. This network was then compared to the similar network created for another archaea, Sulfolobus solfataricus, which is also a well-established archaeal model system (Angelov and Liebl 2006;Snijders et al 2006;Takagi et al 2010;Thürmer et al 2011;Goswami et al 2015). Thus, in the present study, protein-protein interaction networks of chaperones from two archaeal organisms (P. torridus and S. solfataricus) have been compared and subsequently paralleled to a similar network of Homo sapiens (Eukarya).…”