Single molecule electron microscopy provides a powerful approach to study the way in which damaged DNA is remodeled by proteins. The focus of this application is understanding how a number of central human DNA repair and telomere binding proteins interact at large, complex DNA structures containing damage, and how they carry out repair or signal the presence of lesions. This is a highly interactive program which represents longstanding fruitful collaborations with Dr. Paul Modrich working on human mismatch factors, Dr. Aziz Sancar working on human repair signaling factors, and with Dr. Titia deLange working on telomere binding proteins. Together from our own work on this topic and through these collaborations we have published over 20 papers in the past 5 years. This is a highly propitious time to carry out these studies since we have developed two powerful new EM methods: nano-scale biopointers that provide a means of identifying the location of proteins within multi-protein complexes and glycerol spray/low voltage EM that gives a more gentle means of preparing samples for EM. Further, as substrates for these studies, we have produced large natural DNAs containing replication forks or Holliday junctions with nearby mismatched bases and a model telomere DNA. Work on the mismatch repair proteins will take advantage of the recent in vitro reconstitution of nick directed excision repair by the Modrich laboratory. Work on Claspin and the Rad 9- Husl-Radl complex will focus on learning how these proteins interact with replication forks containing damage. Studies of the remodeling of telomeres will take advantage of the recent discovery of discrete multi protein complexes at telomeres. Finally continuing work from our laboratory will focus on p53 as a facilitator of DNA damage recognition. Each system offers a unique window into basic questions of DNA protein remodeling at sites of damage and telomeres and information garnered from one study is immediately applied to the others.
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