Berti Lab: Enzymatic Transition States and Enzyme Inhibitors

Berti Lab

Research

Overview
Antibiotic Targets: AroA & MurA
KIEs by NMR
Training

DNA Repair Enzymes

Our DNA is constantly being damaged - one of the major problems is oxidative damage. The DNA in the human body is 2 x 10¹² m long - it would reach from the Earth to the Sun, and back, over 6 times if it were stretched out in a single, double-stranded helix. Each day, guanine (G) residues are oxidized to 8-oxo-G (OG) about 10¹⁷ times; i.e., every 0.1 mm over its entire two trillion metre length.

OG residues are mutagenic. When OG-containing DNA is replicated, adenine (A) is incorporated with a 200-fold preference over the correct base, C. If the cell divides again, A will pair with T, creating an overall G->T mutation.

We study MutY, an enzyme from E. coli that recognizes OG:A base pair mismatches and catalyzes the first step in the repair pathway. We are interested in MutY works because it is important to maintaining the integrity of the genome. People who are defective in the human equivalent, MYH, get colon cancer in their 40's or 50's.

DNA containing an OG:A mismatch, with the A residue flipped-out, ready to be hydrolyzed by MutY.

The MutY Reaction

MutY hydrolyzes A to adenine and an apurinic (AP) site. In subsequent steps, the AP site is excised, the gap is closed, and OG is repaired.

OG:A mismatch (left) showing the hydrogen bonds involving O8 and N7 of OG(dashed lines). Sites of hydrogen bonding in a normal G:C pair are shown (arrows). (right) Hydrolysis of A to give adenine and apurinic DNA (AP-DNA).

In order to measure KIEs on the MutY reaction, we first synthesize labeled dATP's using radiolabeled glucose, and ¹⁵N-labeled adenine. We then synthesize radiolabeled DNA substrates for MutY and measure KIEs to determine the TS structure.

dATP synthesis. This is a one-pot, 12 enzyme reaction.