Abstract
All cells must maintain their genomic integrity to survive. Several repair mechanisms necessitate unwinding the damaged DNA to allow the recruitment of repair factors. Unwinding activity is achieved by helicases which temporarily destabilize the DNA duplex. When this function fails, human diseases such as Werner Syndrome, Xeroderma Pigmentosum, and Trichothiodystrophy can result. The role of helicases is integral to DNA repair in prokaryotic organisms as well. In Escherichia coli (E. coli), helicases including the RecBCD complex, RecQ, and DinG are utilized to unwind regions of double stranded DNA to make it accessible to repair factors. The SOS response is a system of inducing genes that aid in repairing damaged DNA in E. coli. Though this system has been well characterized, the picture may be incomplete. YoaA is a putative helicase because it shares conserved sequences with damage-inducible helicase DinG and has been shown genetically to be involved in DNA repair. The goal of this study is to develop a system that will allow the purification of YoaA. In our original system, YoaA expression in a high copy plasmid resulted in cell toxicity. In the expression system presented herein, YoaA was fused to a N-terminal RGS-His6 tag (6XHisYoaA) and overexpressed in a low-copy vector under the lacZ promoter. This resulted in a stable form of YoaA. To purify 6XHisYoaA, the Nickel-NTA, single-stranded-DNA, and MonoQ columns were chosen and tested based on the protein’s biochemical and ionic properties. The method that proved to be the most promising in allowing us to purify 6XHisYoaA was utilizing the Nickel-NTA column then the MonoQ column.