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Abstract:

The minichromosome maintenance (MCM) proteins are essential conserved proteins required for DNA replication in archaea and eukaryotes. MCM proteins are believed to provide the replicative helicase activity that unwinds template DNA ahead of the replication fork. Consistent with this hypothesis, MCM proteins can form hexameric complexes that possess ATP-dependent DNA unwinding activity. The molecular mechanism by which the energy of ATP hydrolysis is harnessed to DNA unwinding is unknown, although the ATPase activity has been attributed to a highly conserved AAA+ family ATPase domain. Here we show that changes to N- and C-terminal motifs in the single MCM protein from the archaeon Methanothermobacter thermautotrophicus (MthMCM) can modulate ATP hydrolysis, DNA binding, and duplex unwinding. Furthermore, these motifs appear to influence the movement of the beta-alpha-beta insert in helix-2 of the MCM ATPase domain. Removal of this motif from MthMCM increased dsDNA-stimulated ATP hydrolysis and increased the affinity of the mutant complex for ssDNA and dsDNA. Deletion of the helix-2 insert additionally resulted in the abrogation of DNA unwinding. Our results provide significant insight into the molecular mechanisms used by the MCM helicase to both regulate and execute DNA unwinding.

Abstract:

A DNA methyltransferase activity was identified in a strain of Bacillus thuringiensis that was found to protect DNA from cleavage by the restriction endonuclease HaeIII at overlapping sites. Site-directed mutagenesis was used to confirm therecognition sequence of the methyltransferase as ACGGC.