Abstract
Rev1 is a Y family DNA polymerase that specializes in translesion DNA synthesis. Rev1 is unique in that it preferentially incorporates dCTP in the growing DNA strand, regardless of the templating base. This is because the template base is evicted from the active site and a template amino acid, arginine 324 (R324) acts as the template for the incoming dCTP. We hypothesize that arginine 324 and the neighboring leucine (L325) facilitate the eviction of the DNA template from the active site. To test this hypothesis, we worked to purify R324G/L325G Rev1 double mutant for the purpose of X-ray crystallographic examination of the protein-DNA-dNTP ternary complex. We transformed Escherichia coli (E. coli) and induced expression of both wild type Rev1 and R324G/L325G Rev1. The bacterial cells were lysed by sonification, and the lysate was purified with a GSTrap column. We were able to successfully isolate the Rev1 enzyme. Further purification and crystallization will be necessary to explore the x-ray crystal structure of R324G/L325G Rev1 protein.
Utilization of Escherichia Coli for the growth of Y Family DNA Polymerase Rev1 and GSTrap column for purification
Rev1 is a Y family DNA polymerase that specializes in translesion DNA synthesis. Rev1 is unique in that it preferentially incorporates dCTP in the growing DNA strand, regardless of the templating base. This is because the template base is evicted from the active site and a template amino acid, arginine 324 (R324) acts as the template for the incoming dCTP. We hypothesize that arginine 324 and the neighboring leucine (L325) facilitate the eviction of the DNA template from the active site. To test this hypothesis, we worked to purify R324G/L325G Rev1 double mutant for the purpose of X-ray crystallographic examination of the protein-DNA-dNTP ternary complex. We transformed Escherichia coli (E. coli) and induced expression of both wild type Rev1 and R324G/L325G Rev1. The bacterial cells were lysed by sonification, and the lysate was purified with a GSTrap column. We were able to successfully isolate the Rev1 enzyme. Further purification and crystallization will be necessary to explore the x-ray crystal structure of R324G/L325G Rev1 protein.