THE CRITICAL ACTIVE-SITE AMINE OF THE HUMAN 8-OXOGUANINE DNA GLYCOSYLASE, HOGG1 - DIRECT IDENTIFICATION, ABLATION AND CHEMICAL RECONSTITUTION

Background: Base-excision DNA repair (BER) is the principal pathway re sponsible for the removal of aberrant, genotoxic bases from the genome and restoration of the original sequence. Key components of the BER p athway are DNA glycosylases, enzymes that recognize aberrant bases in the genome and catalyze their expulsion. One major class of such enzym es, glycosylase/lyases, also catalyze scission of the DNA backbone fol lowing base expulsion. Recent studies indicate that the glycosylase an d lyase functions of these enzymes are mechanistically unified through a common amine-bearing residue on the enzyme, which acts as both the electrophile that displaces the aberrant base and an electron sink tha t facilitates DNA strand scission through imine (Schiff base)/conjugat e elimination chemistry. The identity of this critical amine-bearing r esidue has not been rigorously established for any member of a superfa mily of BER glycosylase/lyases. Results: Here, we report the identific ation of the active-site amine of the human 8-oxoguanine DNA glycosyla se (hOgg1), a human BER superfamily protein that repairs the mutagenic 8-oxoguanine lesion in DNA. We employed Edman sequencing of an active -site peptide irreversibly linked to substrate DNA to identify directl y the active-site amine of hOgg1 as the E-NH, group of Lys249. In addi tion, we observed that the repair-inactive but recognition-competent C ys249 mutant (Lys249-->Cys) of hOgg1 can be functionally rescued by al kylation with 2-bromoethylamine, which functionally replaces the lysin e residue by generating a gamma-thia-lysine. Conclusions: This study p rovides the first direct identification of the active-site amine for a ny DNA glycosylase/lyase belonging to the BER superfamily, members of which are characterized by the presence of a helix-hairpin-helix-Gly/P ro-Asp active-site motif. The critical lysine residue identified here is conserved in all members of the BER superfamily that exhibit robust glycosylase/lyase activity. The ability to trigger the catalytic acti vity of the Lys249 --> Cys mutant of hOgg1 by treatment with the chemi cal inducer 2-bromoethylamine may permit snapshots to be taken of the enzyme acting on its substrate and could represent a novel strategy fo r conditional activation of catalysis by hOggl in cells.