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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.

SUBMITTER: Goswami K

PROVIDER: S-EPMC4356968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:DNA methylation events mediated by orphan methyltransferases modulate various cellular processes like replication, repair and transcription. Bacteria and archaea also harbor DNA methyltransferases that are part of restriction-modification systems, which serve to protect the host genome from being cleaved by the cognate restriction enzyme. While DNA methylation has been exhaustively investigated in bacteria it remains poorly understood in archaea. Picrophilus torridus is a euryarchaeon that can thrive under conditions of extremely low pH (0.7), and thus far no reports have been published regarding DNA methylation in this extremophile. This study reports the first experimentation examining DNA methylation in P. torridus. We find the genome to carry methylated adenine (m6A) but not methylated cytosine (m5C) residues. The m6A modification is absent at GATC sites, indicating the absence of an active Dam methylase even though the dam gene has been annotated in the genome sequence. Two other methylases have also been annotated in the P. torridus genome sequence. One of these is a part of a Type I restriction-modification system. Considering that all Type I modification methylases characterized to date target adenine residues, the modification methylase of this Type I system has been examined. The genes encoding the S subunit (that is responsible for DNA recognition) and M subunit (that is responsible for DNA methylation) have been cloned and the recombinant protein purified from E.coli, and regions involved in M-S interactions have been identified. The M.PtoI enzyme harbors all the motifs that typify Type I modification methylases, and displays robust adenine methylation in in vitro assays under a variety of conditions. Interestingly, magnesium is essential for enzyme activity. The enzyme displays substrate inhibition at higher concentrations of AdoMet. Mutational analyses reveal that Motif I plays a role in AdoMet binding, and Motif IV is critical for methylation activity. The data presented here lays the foundation for further research in the area of DNA methylation and restriction-modification research in this most unusual microorganism.

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

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