Artificial processive enzymes based on the T4 replisome

POLY 579

Roeland J. M. Nolte, R.Nolte@science.ru.nl1, Alan E. Rowan, A.Rowan@science.ru.nl1, Stephen J. Benkovic, sjb1@psu.edu2, Joost Clerx, j.clerx@science.ru.nl3, Jeroen JLM. Cornelissen3, Michelle M. Spiering2, and Zhihao Zhuang2. (1) Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, Netherlands, (2) Department of Chemistry, The Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, (3) Department of Organic Chemistry, Institute for Molecules and Materials, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, Netherlands
This project aims to decorate proteins without catalytic activity with catalysts to study the effect of the protein on the catalytic process as a whole. As a model protein, we used the T4 clamp, which ensures the processive replication of DNA by tethering the non-processive DNA polymerase to its substrate in vivo. A cysteine mutant of this protein was expressed in E.coli, purified, and labelled with porphyrins and Fe-EDTA complexes. Porphyrins are able to complex in the minor groove and cut at AAA sites when activated, while the iron complex will degrade DNA non-specifically under DNA footprinting conditions. The activity of the modified proteins were tested and compared to the wild type system, and used for the oxidation of DNA substrates. The processive nature of this catalytic process was investigated. Futhermore, application of these artificial enzymes for the epoxidation of synthetic double bond containing polymers will be discussed.

 

Biocatalysis in Polymer Science
8:30 AM-11:45 AM, Wednesday, 13 September 2006 San Francisco Marriott -- Salon 12/13, Oral

Division of Polymer Chemistry

The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006