WHAT LEVEL OF GENETIC FIDELITY DO HYPERTHERMOPHILIC ARCHAEA MAINTAIN (AND HOW DO THEY DO IT)?            [Grogan lab]

Each nucleotide in an organism’s genome has a finite probability of undergoing spontaneous chemical decay before the next round of DNA replication. This probability reflects several temperature-dependent chemical reactions (depurination, deamination, hydrolysis, alkylation, and oxidation), and it is about a thousand-fold higher at 80ºC than at 37ºC. This, combined with our observations that diverse forms of DNA damage all induce mutation in S. acidocaldarius, allows a clear prediction to be made: either (A) S. acidocaldarius has efficient mechanisms that compensate for the high rate of DNA damage promoted by its growth temperature, or (B) S. acidocaldarius has a much higher rate of spontaneous mutation than mesophilic micro-organisms have.

We have validated the use of 5-fluoro-orotic acid (FOA) to assay loss-of-function mutations in S. acidocaldarius and have shown that the aggregate rate of mutations inactivating either the pyrE or pyrF genes is about 3 x 10^-7 events per cell division. When normalized for the sizes of both genes, the rate closely matches those measured for protein-encoding genes of E. coli [publication 4]. Collaboration with Dr. John Drake at the NIEHS (Research Triangle Park, NC) resulted in a more detailed analysis based on the mutational spectrum of the S. acidocaldarius pyrE and pyrF genes [publication 14]. Most of the mutations occurred in pyrE, and base-pair substitutions were remarkably rare (diagram below). The corresponding genomic rate of mutation is among the lowest yet calculated for microbial DNA genomes ranging from the extremely small (bacteriophage M13) to the large (Neurospora crassa).

Although these results clearly support alternative (A) above, they do not indicate what gene products enforce this genetic fidelity, or how they work. Ironically, the completely sequenced genomes of hyperthermophilic archaea lack genes for the mismatch-repair proteins that are otherwise highly conserved in all other organisms and required for their low spontaneous mutation rates [reviewed in publication 12].