Analysis of single molecules by EM provides a powerful approach to study the mechanics of DNA replication. The long-term focus of this study combines biochemistry, protein tagging, with direct visualization of DNA protein complexes by electron microscopy (EM). A statistically large number of molecules are examined and this information is combined with data from biochemical assays. This project has great strength in its long standing interactions with Dr. Steve Bell (yeast ORC), Drs. Tom Broker and Louise Chow (human papillomaviruses (HPV)), and Dr. Charles Richardson (T7 replication). These studies are augmented with work carried out in this laboratory on T4 and Herpes Simplex type 1 (HSV-1). The goal is to understand origin activation (HPV, yeast) and the architecture of a moving fork (T7, T4, HSV-1). A new technology using nano-scale paramagnetic particles attached to specifically designed DNA templates allows visualization of nanogram quantities of DNA and protein and the rapid exchange DNA-protein complexes into buffers optimal for high resolution EM. DNAs containing the yeast ARS1 element attached to magnetic particles will be incubated with yeast cell extracts to load the pre-RC complex including Mcm and Cdc6 factors. Their structure will be probed by Western analysis and EM using nanoscale 'biopointers'. DNA fragments containing the HPV origin bound to paramagnetic particles will be assembled with the E1, E2 proteins as well as factors from 293 cell extracts. The stepwise opening of the origin and its interaction with cell factors including p53 will be investigated using a combination of EM, Western analysis, and replication assays. Continued studies of the moving fork in T7 and T4 will employ paramagnetic particles along with single particle image reconstructions and cryoEM to generate high resolution structures containing the polymerase, helicase-primase, SSBs and other factors on model replication forks. Using HSV-1 replication proteins purified in this laboratory, the architecture of DNA-protein complexes which catalyze replication will be examined to provide information on the looping of the lagging strand in a moving eukaryotic fork. Using these in vitro T4 and HSV-1 replication systems, the way in which recombination coupled replication is initiated by recombination factors and how the replication forks proceed to generate networks of replicating DNA will be visualized. Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the development of drugs which bind key components at replication forks. This will help lead to development of new anti-bacterial, fungal, and viral compounds.
Understanding the structure of the proteins and protein complexes which catalyze replication of prokaryotic and eukaryotic chromosomes and viruses is crucial to the rational design of new drugs which bind these proteins and structures. This study employs high resolution electron microscopy to provide a unique and direct visualization of protein complexes at replication origins and replication forks in bacteria, fungi, eukaryotic cells and viruses to provide the information needed to identify new drug targets.
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