Expression and Characterization of YoaA, a Putative Helicase in Bacteria, Involved in Repairing Blocks to DNA Replication

Abstract

While cells have efficient pathways for repairing damage to DNA, some DNA damage avoids repair and is found in DNA replication. Because the DNA polymerases that replicate the genome are high fidelity enzymes, DNA damage blocks DNA synthesis and ultimately progression of the replication fork. It is known that cells would not be able to survive without mechanisms to repair these replication blocks and to restart replication. With a genetic screen, our collaborators at Lovett laboratory, using 3’azidothymidine (AZT) as a tool to inhibit replication, identified a novel gene in Escherichia coli, yoaA, which was required along with holC to provide cells with tolerance to AZT. HolC is a protein subunit of the E.coli DNA polymerase III holoenzyme that does the bulk of synthesis during DNA replication, and yoaA binds HolC. Based on sequence, the yoaA gene encodes an iron-sulfur (Fe-S) helicases. E.coli contains a second Fe-S helicase, DinG, and human cells contain four Fe-S helicases, XPD, FANCJ, RTEL1, and ChlR1, that are involved in DNA repair. The overall goal of this project is to express YoaA protein in soluble form and characterize its biochemical activities to determine how YoaA aids in DNA replication fork repair. In our initial studies, we made fluorescent labeled DNA for helicase assays and tested them which allowed us to work towards our long term goals of determining: 1) whether YoaA is DNA helicase and what are the best substrates for YoaA, and 2) how HolC affects YoaA activities. We have subcloned the yoaA gene into different expression vectors to express YoaA with and without affinity tags, and to co-express YoaA with HolC. We are developing strategies to purify YoaA alone and in a complex with HolC. We have subcloned the yoaA gene into different expression vectors to express yoaA with pCOLADuet. We are developing strategies to purify YoaA alone. The initial results working towards this are presented.