A41
Can the human minichromosome maintenance proteins form a processive helicase?
Richard Parker, Rubab Satti, James Chong
University of York, York, UK
Minichromosome maintenance (MCM) genes are up-regulated in cancer cells. Our aim is to better understand how the MCM proteins function in order to provide potential targets for cancer therapy. MCMs are essential for the initiation and progression of DNA replication in proliferating cells. It is hypothesised that the hexameric MCM2-7 complex unwinds duplex DNA at replication forks. However, helicase activity has only been demonstrated in a sub-complex of MCMs (4, 6 and 7) and only with a limited degree of processivity. Our goal is to reconstitute the complete human MCM complex from recombinant proteins and demonstrate a processive helicase activity. We intend to dissect this activity further by incorporating mutations into the recombinant complex to ascertain the functional contribution of each subunit. We have chosen to use a bacterial expression system to avoid potentially inhibitory post-translational modifications and the possibility of forming mixed exogenous / endogenous MCM complexes. We have coexpressed the six MCM subunits in E. coli. By using affinity tag pull-down assays and western blotting we have shown that all six proteins coprecipitate from E. coli extracts, suggesting the formation of a complete complex. We are currently developing a purification protocol to isolate this complex from E. coli cell lysates so that it can be tested for helicase-associated activities. We hope that this recombinant human MCM system will enable us to gain a better understanding of molecular mechanisms associated with MCM helicase activity, and ultimately provide insight into the role of the human MCM complex in cancer.
