THERMOSTABLE ARCHAEAL O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASES

Archaea represent some of the most ancient organisms on earth, and the y have relatively uncharacterized DNA repair processes. We now show, u sing an in vitro assay, that extracts of two Crenarchaeota (Sulfolobus acidocaldarius and Pyrobaculum islandicum) and two Euryarchaeota (Pyr ococcus furiosus and Thermococcus litoralis) contain the DNA repair pr otein O-6-alkylguanine-DNA alkyltransferase (ATase). The ATase activit ies found in the archaea were extremely thermostable, with half-lives at 80 degrees C ranging from 0.5 hr (S, acidocalrlarius) to 13 hr (T. liroralis), The temperature optima of the four proteins ranged from ap proximate to 75 to approximate to 100 degrees C, although activity was seen at 37 degrees C, the temperature optimum of the Escherichia coil and human ATases. In all cases, preincubaton of extracts with a short oligonucleotide containing a single O-6-methylguanine residue caused essentially complete loss of ATase activity, suggesting that the alkyl phosphotriester-DNA alkyltransferase activity seen in some prokaryotes is not present in Archaea. The ATase from Pyrobaculum islandicum had an apparent molecular mass of 15 kDa, making it the smallest of these proteins so far described. In higher organisms, ATase is responsible f or the repair of toxic and mutagenic O-6-alkylguanine lesions in alkyl ated DNA, The presence of ATase in these primitive organisms therefore suggests that endogenous or exogenous exposure to agents that generat e appropriate substrates in DNA may be an early event in evolution.