Jon K. De Riel, PhD
Assistant Professor, Fels Institute for Cancer Research and Molecular Biology
- Contact Information
- About Me
Our longstanding collaboration with Dr. Earl Henderson (Fels Institute) is focused on the molecular biology of the denV gene from bacteriophage T4, which encodes endonuclease V, a 16 kDa DNA repair protein that catalyzes two distinct steps in the base excision repair of pyrimidine dimers. Since 1984, we have extensively characterized the biology of den V-mediated UV repair in both prokaryotic and eukaryotic cells. Because endonuclease V possesses two distinct catalytic activities (apyrimidine dimer-DNA glycosylase activity and an apyrimidinic-site endonuclease) in a single small structural domain, its mechanism is of considerable interest. For structure-function analysis of the protein, we have developed a method of random targeted mutagenesis, based on a previously published procedure for polymerase chain reaction mutagenesis that can be used to blanket a selected small region of the gene with random mutation. The resulting mutations are then identified by sequencing and correlated with the catalytic properties of the corresponding altered proteins. The method is useful because it allows random targeted mutagenesis to be performed directly in a plasmid expression vector, permitting immediate functional screening of the resulting mutants. This procedure has been used to analyze the functional importance of the Ala(116)-Lys(121) region of endonuclease V. Screening of the resulting clones detected a high percentage of mutants (19/22) which included 15 unique mutants. Analysis of the UV-repair properties of these mutants, and studies of purified endonuclease V from them, show an important role for this region in substrate binding, and also suggest a direct correlation between UV survival and both DNA binding and pyrimidine dimer-DNA glycosylase activity, but not apyrimidinic-site endonuclease activity. Current efforts are concerned with extending random targeted mutagenesis studies to other regions of the molecule, with the intent of separating the two catalytic activities into two different modified proteins; and also examining the effects of the den V gene on mutagenesis in human cell lines.
- Education, Training & Credentials
- Harvard Medical School, Boston, MA