Biochemical characterization of the Minichromosome maintenance (MCM) helicase from Methanothermobacter thermautotrophicus

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DNA replication requires coordination of numerous proteins to duplicate genetic information in a precise and timely manner. One of the key players in replication is the replicative helicase that unwinds the duplex DNA to provide the single-stranded template for the DNA polymerases. Minichromosome maintenance (MCM) protein is the replicative helicase in archaea. This dissertation focuses on the MCM helicase from the euryarchaeon Methanothermobacter thermautotrophicus (Mth).

 Archaeal MCM proteins can be divided into two major parts, the N terminal and C terminal domains.  The N terminal domain is essential for DNA binding and multimerization, while the C-terminus contains the catalytic domains.   The objective of this dissertation is to elucidate the mechanism by which the N terminal domain communicates with the catalytic domain to facilitate helicase activity.   To address this question, two approaches were taken.  One approach identified conserved residues found in the N terminus and investigated their properties using various biochemical and biophysical methods.  By analyzing several proteins with mutations in the conserved residues, a loop that is essential for MCM helicase activity was identified.  The study suggests that the loop is involved in coupling the N-terminal DNA binding function and the catalytic activity of the AAA+ domain.  Some other conserved residues, however, did not directly affect the MCM helicase activity but showed differences in biochemical properties suggesting that they may play a role in maintaining the structural integrity of the MCM helicase.  Another approach determined the differences in thermal stability of the MCM protein in the presence of various cofactors and DNA substrates.  The study shows that the protein has two unfolding transitions when ATP and the DNA are present, while non-hydrolyzable ATP results in one transition.  This study suggests possible conformational changes arising from decoupling of two domains that occur during the ATP hydrolysis in the presence of DNA.  Furthermore, both DNA binding function by the N terminal domain and ATP binding by the catalytic domain are essential for the change.