Abstract
Helicases are DNA binding proteins that bind to a DNA strand during synthesis, repair, and translation16. These proteins allow for the two strands of DNA to become separated into single strands which can then be processed by a DNA polymerase or other DNA machinery16. DinG is a helicase that is a member of the helicase superfamily 216. These helicases use ATP to complete their functions and contain an iron sulfur cluster and transcribe in the 5’-3’ direction on a DNA strand3,16. YoaA is a paralog to DinG and could exist within the same family of helicases16. YoaA is a putative DNA helicase that becomes active in Escherichia Coli (E. Coli) in response to DNA damage and takes part in the DNA damage response3. All cells must replicate their DNA with high fidelity, however single stranded DNA breaks occur regularly during replication17. These single stranded breaks can become double stranded breaks without repair and the cell has developed a specialized suite of DNA damage proteins to complete these breaks1. HolC is a part of the clamp loader complex found at the replication fork that binds to YoaA and allows it to carry out its function1. Cells without YoaA and HolC functionality have been shown to have greater issues addressing damage from DNA damaging agents2. Specifically, YoaA has been shown to confer resistance to Azidothymidine (AZT), a known DNA damaging agent8. Investigated herein is a YoaA-6xHIS construct in an overexpression plasmid that was used to investigate YoaA in the context of its response to mutation. Mutants in an over expression plasmid were investigated by Western Blot and subsequent immunoblotting. In addition, YoaA constructs utilizing HA, Venus, Flag, 3xFlag and 6xHIS tags were P1 transduced in a native MG1655 cells and placed on the chromosome. These wildtype constructs were then used to investigate YoaA expression levels in a native cell type. Cultures of these tagged constructs were grown in the presence of AZT in the hopes of inducing YoaA overexpression as a method of inducing DNA damage. We did see proof that mutation of the HolC binding domain in YoaA did not cause any significant changes in expression levels of the YoaA mutants we investigated using an over expression plasmid. However, we were not able to get the chromosomal constructions to express detectable levels of YoaA protein if YoaA was induced.