The purpose of this research is to further the understanding of DNA damage and repair by elucidating the relationship between these processes and DNA nanomechanics and by visualizing repair protein activities on DNA using atomic force microscopy techniques. The objectives are: 1) optimize the AFM platform for single-molecule DNA measurements 2) examine various lesions in individual DNA molecules and follow in the AFM their direct reversal by DNA repair enzymes 3) use AFM imaging and force spectroscopy to visualize the mismatch repair reaction of E. coli. To achieve these objectives the AFM platform needs to be optimized to reduce force errors to single piconewtons and to accelerate image acquisition by using the ultra-small cantilever technology. DNA repair proteins such as photolyases, endonucleases and ligases will be used as damage markers and will be imaged in complexes with DNA by AFM in order to locate and identify the damage sites. Force spectroscopy measurements will determine the mechanical fingerprints of various types of DNA damage caused by UV and gamma radiation and will directly follow damage reversal in the presence of the repair activities. This project will also examine and visualize the DNA mismatch repair reaction using E. coli repair activities and AFM imaging and force spectroscopy technologies. This project will provide an exciting education and research opportunity for three graduate students at the interface between engineering and biology and will likely develop new ultra sensitive assays for DNA damage detection that is of significance to many areas of biology.
