Abstract: Series 107, Lecture 5

The Harvey Lectures Series 107 (2011—2012)

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Lecture #5: Thursday, March 15, 2012 — Time and Location

Watching Individual Proteins Working on Single Molecules of DNA: From Biophysics to Cancer

Stephen C Kowalczykowski, PhD

Stephen C Kowalczykowski, PhD

Distinguished Professor of Microbiology and of Molecular & Cellular Biology

University of California, Davis

Davis, California

Dr Kowalczykowski's Website

DNA breaks are a chronic and ubiquitous type of DNA damage. The homology-directed repair of these breaks is a multi-step process requiring scores of proteins that act in concert to re-establish the broken DNA connection. We can now visualize the action of many of these proteins at the single-molecule level. This is achieved by either optical trapping, or surface attachment of an individual DNA molecule. Protein, or protein action, is detected by direct imaging of fluorescent proteins. Individual molecules are visualized using epifluorescence microscopy or total internal reflection fluorescence (TIRF) microscopy. We have imaged translocation of DNA motor proteins and self-assembly of DNA strand exchange proteins. In parallel, we biochemically reconstituted the DNA-break repair machinery from human cells. These proteins mediate DNA processing, homologous pairing, and final separation of repaired chromosomes. Alteration of these processes leads to chromosome instability and cancer. The functions of BLM, DNA2, and MRE11-RAD50-NBS1 in processing; BRAC2 in RAD51-mediated DNA pairing; and BLM and Topoisomerase III in topological dissolution will be summarized. The importance of seeing individual molecules at work, and the connections between single-molecule biophysics and the molecular etiology of breast cancer will be elaborated visually.