DNA is known as the carrier of all genetic material for living organisms. Although DNA is generally stable, it can be damaged when exposed to chemicals in the environment. Even addition of a single carbon atom to a DNA base poses a serious threat to cells because it can cause genetic mutations or cell death. Cells have evolved many pathways to combat DNA damaging chemicals, including the base excision repair pathway that begins with a glycosylase enzyme. Glycosylase enzymes remove damaged alkylated bases from DNA. In bacteria, the main glycosylase is called AlkA while human cells have the AAG glycosylase. This differs in the case of Bacillus subtilis, a bacterium commonly found in soils and the human gastrointestinal tract. Interestingly, B. subtilis contains AlkA, AAG and another proposed alkylation repair glycosylase, YfjP. It is not understood why B. subtilis has extra glycosylase enzymes, and whether they act in the same manner as their counterparts in other bacteria and humans. We successfully purified the WT AAG and AlkA glycosylase using affinity and ion exchange chromatography. Additionally, we also purified a YfjP fusion construct. We are studying these enzymes with kinetic assays to understand why B. subtilis contains a canonical eukaryotic protein. Kinetic experiments are being run to determine the speed of each mechanistic step. We will test the specificity of each enzyme with various DNA bases including N3-methylcytosine, N1-methyladenine, 1,N2-ethenoguanine, hypoxanthine, and 1,N6-ethenoadenine.

Purifying and characterizing Bacillus subtilis DNA repair glycosylase enzymes

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