Conversion of the bifunctional 8-oxoguanine/β-δ apurinic/apyrimidinic DNA repair activities of Drosophila ribosomal protein S3 into the human S3 monofunctional P-elimination catalyst through a single amino acid change

The Drosophila 53 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that 53 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila 53 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a beta -elimination reaction that cleaves phosphodiester bonds 3' and adjacent to an AP lesion in DNA. In certain situations, this is followed by a delta -elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5' and 3'-phosphate groups. The human 53 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a beta -elimination reaction, thus further distinguishing itself from the Drosophila 53 protein in lacking a delta -elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human 53 were suggested by the absence of a conserved glutamine residue in human 53 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the delta -elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human 53 protein. In support of this were experiments executed in vivo that showed that human 53 and the Drosophila site-directed glutamine-changed 53 performed poorly when compared with Drosophila wildtype 53 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.