Microbes whose genomes are encoded by DNA and for which adequate information is available display similar genomic mutation rates (average 0.0034 mutations per chromosome replication, range 0.0025 to 0.0046). However, this value currently is based on only a few well characterized microbes reproducing within a narrow range of environmental conditions. In particular, no genomic mutation rate has been determined either for a microbe whose natural growth conditions may extensively damage DNA or for any member of the archaea, a prokaryotic lineage deeply diverged from both bacteria and eukaryotes. Both of these conditions are met by the extreme thermoacidophile Sulfolobus acidocaldarius. We determined the genomic mutation rate for this species when growing at pH 3.5 and 75°C based on the rate of forward mutation at the pyrE gene and the nucleotide changes identified in 101 independent mutants. The observed value of about 0.0018 extends the range of DNA-based microbes with rates close to the standard rate simultaneously to an archaeon and to an extremophile whose cytoplasmic pH and normal growth temperature greatly accelerate the spontaneous decomposition of DNA. The mutations include base pair substitutions (BPSs) and additions and deletions of various sizes, but the S. acidocaldarius spectrum differs from those of other DNA-based organisms in being relatively poor in BPSs. The paucity of BPSs cannot yet be explained by known properties of DNA replication or repair enzymes of Sulfolobus spp. It suggests, however, that molecular evolution per genome replication may proceed more slowly in S. acidocaldarius than in other DNA-based organisms examined to date.A rchaea isolated from geothermal environments grow optimally at temperatures that are lethal to all genetically well characterized microorganisms and damaging to DNA. The enzymes of these hyperthermophilic archaea are intrinsically thermostable due to a variety of structural features that discourage protein unfolding, which explains how metabolism can be maintained at extremely high temperatures (1). The strategy of intrinsic stabilization does not seem to apply to the chromosomes of these archaea, however, and does not address the problem of spontaneous DNA decomposition at physiological temperatures (2). Although information about the gene content, genome organization, and evolutionary divergence of hyperthermophilic archaea is expanding rapidly, their basic genetic processes remain largely unexplored. As a result, it is unclear how these organisms compare with well studied microbes with respect to genetic exchange, DNA repair, mutation, genetic exchange, and other fundamental processes important to their survival and evolution.Rates of spontaneous mutation measured in microbial systems provide evidence of the biological importance of genetic fidelity. Accurate rates of spontaneous mutation per genome are available for only six DNA-based microbes: phage M13, phage , phage T2͞T4, the bacterium Escherichia coli, the yeast Saccharomyces cerevisiae, and the filamentous...

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase ␣ class) enzymes that determine the fidelity of phage DNA replication. A T4 whose gene 43 has been mutationally inactivated can be replicated by a cognate RB69 gp43 encoded by a recombinant plasmid in T4-infected Escherichia coli. We used this phage-plasmid complementation assay to obtain rapid and sensitive measurements of the mutational specificities of mutator derivatives of the RB69 enzyme. RB69 gp43s lacking proofreading function (Exo ؊ enzymes) and/or substituted with alanine, serine, or threonine at the conserved polymerase function residue Tyr 567 (Pol Y567(A/S/T) enzymes) were examined for their effects on the reversion of specific mutations in the T4 rII gene and on forward mutation in the T4 rI gene. The results reveal that Tyr 567 is a key determinant of the fidelity of base selection and that the Pol and Exo functions are strongly coupled in this B family enzyme. In vitro assays show that the Pol Y567A Exo ؊ enzyme generates mispairs more frequently but extends them less efficiently than does a Pol ؉ Exo ؊ enzyme. Other replicative DNA polymerases may control fidelity by strategies similar to those used by RB69 gp43.Bacteriophage RB69 is a relative of phage T4, with which it shares many similarities in genetic organization (1, 2) and structures and functions of the phage-encoded DNA replication proteins (3,4). Replication fidelity in T4 and presumably also in RB69 is determined almost exclusively by the fidelities of the phage-encoded DNA polymerase and its associated proofreading 3Ј-5Ј exonuclease (5). This useful simplicity reflects the fact that T4 DNA replication appears to be devoid of DNA mismatch repair; phage T4 is not subject to the action of the several Escherichia coli mismatch repair systems (6) and seems unable to repair mutational heteroduplexes on its own. Screens for T4 mutator mutations have failed to uncover evidence for the involvement of mismatch repair in mutagenesis, and the mutational dose response to base analogues does not display the mismatch repair-dependent lag seen in E. coli (5).The DNA polymerases of phages T4 and RB69 (gp43, product of phage gene 43) are members of the polymerase ␣ class or B family of DNA polymerases, which includes the replicative polymerases ␣, ␦, and ⑀ of eukaryotic cells and the polymerases of several of their DNA viruses (7). Some archaeons also encode gp43-like B family enzymes (8 -10). As such, T4 gp43 and RB69 gp43 are attractive subjects for studies of mechanisms of replication by this class of enzymes, particularly because of the amenability of the phage system to combined genetic and biochemical analyses (11)(12)(13)(14). A recently determined crystal structure of RB69 gp43 reveals five discrete domains termed N, Exo, Palm, Fingers, and Thumb (15). This structure is in the "open" configuration and provides a preliminary framework for understanding the dynamics of DNA polymerase interactions with the DNA primer template, with incoming dNTPs, and ...

Spontaneous mutations in the orotate:phosphoribosyl transferase (pyrE2) gene of the halophilic archaeon Haloferax volcanii were selected by 5-fluoroorotic acid plus uracil at a rate of $2 3 10 À8 /cell division in fluctuation and null-fraction tests but $6 3 10 À8 /cell division in mutation-accumulation tests. The corresponding genomic mutation rates were substantially lower than those observed for other mesophilic microbial DNA genomes on the basis of similar target genes. The mutational spectrum was dominated by indels adding or deleting multiples of 3 bp. Properties of the organism contributing to this unusual mutational pattern may include phenotypic lag caused by a high chromosomal copy number and efficient promotion of strand misalignments by short direct repeats.A NALYSES of spontaneous mutation in diverse micro-organisms have provided important insights into the fundamental forces and molecular mechanisms determining genetic fidelity. In most of these studies, a selection is used to quantify the rate of forward (i.e., inactivating) mutations in one or more chromosomal genes. Sequencing of representative mutants then reveals a spectrum of mutation, which enables the efficiency of mutation detection and other parameters to be estimated. Such analyses have shown that all mesophilic micro-organisms examined (including DNA viruses) share two mutational characteristics: (i) the rates of mutation per genome fall near 0.003/replication, despite large differences in mutation rates per base pair (Drake 1991;Drake et al. 1998;Drake and Hwang 2005), and (ii) $70% of the observed mutations are base-pair substitutions (BPSs) (Grogan et al. 2001). However, extending this analysis to an archaeon from a geothermal environment revealed an apparently lower genomic rate (#0.0018) and a lower proportion of BPSs (33%) (Grogan et al. 2001). These results suggest that basic mutational properties of micro-organisms may adapt to unusual environmental conditions.Halophilic archaea grow optimally at moderate temperatures in 1-4 m salt (Rodriquez-Valera 1995). They cope with these environments by maintaining high intracellular concentrations of potassium and chloride (Lanyi 1974), which can be expected to exert molecular stresses on genetic processes. Certain genetic methods have been developed for Halobacterium salinarum and Haloferax volcanii (Allers and Mevarech 2005; Soppa 2006), and various systems for repairing DNA damage have been reported in these two species (McCready 1996;Baliga et al. 2004;Kottemann et al. 2005), but neither halophile has been evaluated for the accuracy of genome replication. In this study, we demonstrated the ability of 5-fluoroorotic acid (FOA) to select spontaneous orotate:phosphoribosyl transferase (OPRTase) mutants of H. volcanii. We measured the rate of spontaneous mutation in the corresponding pyrE2 gene, analyzed a number of mutants by DNA sequencing, and evaluated the implications for genomic mutation in this organism.The H. volcanii wild-type strain DS70 (Wendoloski et al. 2001) was cul...