The Andrew Bohm Lab
Reprogramming CRE
Cre recombinase has been used to edit DNA in vivo and in vitro for decades. The Cre protein forms a tetramer on DNA that can insert, delete, or invert large DNA sequences with single-nucleotide precision. In recent years, genetic selection schemes have allowed Cre’s specificity to be altered. Working with Frank Buchholz (University of Dresden) we have determined the crystal structure of a reprogrammed Cre that targets the long terminal repeats (LTRs) that flank the genes of HIV after the retrovirus has inserted itself into the genome of host cells. We are now using this structure, along with a growing database of Cre mutations that alter specificity, to streamline the process of engineering new, highly potent versions of the recombinase.
Figure 1. Crystal structure of Tre in complex with a DNA fragment from the HIV LTR. Tre contains 19 amino acid substitutions (red spheres) relative to Cre. Its DNA target differs at 17 of 34 positions relative to LoxP, the sequence targeted by Cre.
Structural Biology of DNA Tumor Viruses
Despite their small genomes (~5kb) and their small repertoire of encoded proteins, polyomaviruses are potent in their ability to transform infected cells. We have worked for over a decade on the nucleic acid recognition that regulates polyomavirus replication at the viral origin. Our current work in this area focuses on the interaction of small T-antigen with protein phosphatase 2A. This work is a collaboration with Brian Schaffhausen (Tufts University). Understanding the details of this interaction will help us understand why some polyomaviruses are more potent than others with respect to cellular transformation and how isolated T-antigen proteins are able to transform cells and influence their differentiation.