Atomic force microscopy studies of DNA binding properties of the archaeal MCM complex

The MCM (mini-chromosome mainteinance) protein complex has a key role in the replication machinery of Eukaryotes and Archaea. Beside the main unwiding role, this helicase is also supposed to act as one of the essential element licensing replication to ensure that each segment of the genome is replicated only once per cycle. Six homologous MCM proteins belonging to the AAA+ ATPase superfamily, known as MCM2-7, are forming in Eukaryotes a hexameric hetero-complex that is supposed to "load" onto a single DNA strand and to use energy from ATP (Adenosine triphosphate) hydrolysis to unzip the DNA double helix. In Archaea, there is a single MCM gene, coding for a single MCM protein. Therefore the protein complex is made by a homohexamer with six equivalent subunits. The DNA loading mechanism for double-strand unzipping is similar to the Eukaryotes case. The scope of this thesis work is to investigate the structural details of the interaction between DNA and the MCM complex in near physiological condition, by means of Atomic Force Microscopy (AFM) single molecule imaging. For that, we used archaeal MCM from Methanothermobacter Thermautotrophicus as a model system. There are structural and biochemical evidences that MCM interacts with DNA in two ways: the canonical “loaded” mode where the MCM protein complex is encircling the DNA for unwinding, and an “associated” mode where the DNA is supposed to be wrapped around the external part of the proper ring structure. In this second configuration, less studied than the loaded one, the binding interaction between MCM and DNA might play a role in licencing/initiating the replication process. By means of AFM, we studied the conformational changes induced by the protein complex on blunt-ends, double-stranded (ds) DNA filaments of different lenghts. AFM experiments were carried out in two different conditions: in air to understand the static conformations of DNA-protein complexes; in liquid to follow the interaction dynamics. We first optimized the protocol (surface treatment, buffer condition) for AFM imaging in air to obtain high resolution images of surface-equilibrated DNA molecules before and after the interaction with the protein complex. From statistical analysis of AFM images, we localized the protein complexes along the isolated dsDNA sequences and calculated DNA contour lenght and bending angle before and after the interaction with the protein complex. We discriminated between proteins with DNA wrapped around, calling them “associated”, and the ones interacting with DNA without inducing any bending, calling them “loaded”. To confirm this topographical assignment, we tested two mutants: NβH complex, which presents a mutation in the central hole of the hexamer, inhibiting DNA loading; ∆sA complex, which presents a mutation in the external part, preventing DNA association. In the case of NβH, only associated DNA was observed; in the case of ∆sA, only loaded complexes were found, to prove the soundness of our assumptions. Moreover, the total number of DNA bound complexes decreased from 84% to 20% from MCM to ∆sA, proving that association is involved in favouring the replicative helicase loading, and initiating the double-helix unwinding. Finally, we found a DNA compaction of about 13 nm for wild type MCM and ∆sA mutant. In the case of NβH mutant, the compaction is of about 18nm, and comes together with a bending angle increase of about 16°, strongly supporting the “association” model. Finally, we studied the dynamics of DNA-MCM complex interaction, in the presence of ATP, via moderately fast (few seconds/images) AFM imaging in liquid. After ATP loading, we observed a change of topographic height of the DNA strands, consistent with the formation of ssDNA, as a sign of DNA unwinding by the MCM complex. This effect was unexpected, since there are no biochemical evidences in literature of efficient unwinding of MCM complex on blunt-ended DNA.