Human DNA may be routinely damaged by many mechanisms including chemical carcinogens and reactive oxygen molecules occuring naturally in the atmosphere. We are using a site specific approach for defining structure-function relationships with the long range goal of understanding how DNA damage is recognized by repair enzymes using DNA containing 8-oxoguanine (8oxodG), and FPG protein from E. coli as our prototype system. We have established that a single zinc finger motif is involved in binding FPG protein to DNA duplexes containing 8-oxoguanine. We are currently trying to determine the structure of this complex by NMR spectroscopy and x-ray crystallography. Recently, we have cloned a mammilian FPG protein that performs the same function as the E. coli FPG but it is a completely different protein. We have also obtained crystals for this mammilian protein. Concurrently, we are applying site-directed mutagenesis techniques to the zinc finger motif portion of the FPG protein guided by molecular modeling studies. These studies include extensive molecular mechanics and molecular dynamics calculations carried out using the X-PLOR program.
We have completed a preliminary molecular model of the FPG zinc finger - DNA complex with initial molecular dynamics simulations (see image above or download this high resolution tiff image [1.7MB]). We are currently running simulations of this complex with full solvation and ions. The results of our initial molecular dynamics simulations support the hypothesis that there is a correlation between the amino acid sequence of a DNA recognizing protein. We have taken into account sequence homologies between FPG proteins from a number of organisms and the consistency of the pattern of intermolecular hydrogen bonds between the FPG protein and its complexed DNA lend credibility to the veracity of the proposed structure as design by analogy techniques.
Acknowledgments: This research is supported by the National Institutes of Health, grant R01-CA17395 (A.P. Grollman, P.I.), the National Cancer Institute, grant P01-CA47995 (A.P. Grollman, P.I.), and the National Center for Research Resources, grant P41-RR01081 (T.E. Ferrin, P.I.). Molecular models were created using the suite of programs available in UCSF MidasPlus.
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