An integrated structural and biochemical approach to human DNA replication
The capacity to maintain and transmit the genetic information from one generation to the next relies entirely on the accuracy and regulation of the mechanisms of DNA replication. The problem is particularly serious for the more complex eukaryotes, which contain much more genetic information than simple bacteria. In these organisms DNA replication has to start at multipleorigins along the genome and a complex network of proteins, under strict cell-cycle control, is required to ensure that each origin is used only once and no segment of DNA is left un-replicated or undergoes multiple rounds of replication.
We are using a variety of structural biology techniques to elucidate the structure of various factors involved in this process in human cells. Our target include a number of DNA helicases (such as the MCM complex, RecQ1 and RecQ4), the human DNA primase heterodimer, and many accessory factors (such as GINS, Cdc45, MCM10, AND-1). Although our main aim is to produce crystals suitable for macromolecular crystallography, we are also using NMR on small domains, as well as low resolution techniques such as electron microscopy and small-angle X-ray scattering to visualize the overall architecture of large macromolecular complexes and/or flexible molecules. The structural studies are complemented by biochemical and biophysical experiments.
Misregulation of the DNA replication process has been found to occur frequently in cancer cells. Moreover, most of these proteins are only found in actively growing cells, and are therefore potential proliferation markers and possible drug targets for anti-cancer drugs.