<i>Escherichia coli</i> Orf135 (NudG) mutant protein specific for oxidized dATP

Damaged 2'-deoxyribonucleotides cause mutations, cancer, cell death, and aging. The <i>Escherichia coli</i> Orf135 (NudG) protein catalyzes the hydrolysis of various 2'-deoxyribonucleotides including an oxidized form of dATP, 2-oxo-1,2-dihydro-2'-deoxyadenosine 5'-triphosphate (dA^OTP, 2-hydroxy-2'-deoxyadenosine 5'-triphosphate). The best substrate is 5-methyl-2'-deoxycytidine 5'-triphosphate (dC^mTP), and the protein prefers dC^mTP over dA^OTP by ∼200-fold in vitro. To make the enzyme specific for the mutagenic nucleotide dA^OTP, a double mutant protein (E33A plus D118E) was designed and produced in <i>E. coli</i>. The purified mutant protein showed one order of magnitude higher dA^OTP preference over dC^mTP. The split protein based on this mutant may potentially be used to detect dA^OTP in living cells.