Genetic fidelity under harsh conditions: analysis of spontaneous mutation in the thermoacidophilic archaeon Sulfolobus acidocaldarius.

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 degrees 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.

Conditional coupling of leading-strand and lagging-strand DNA synthesis at bacteriophage T4 replication forks.

Eight proteins encoded by bacteriophage T4 are required for the replicative synthesis of the leading and lagging strands of T4 DNA. We show here that active T4 replication forks, which catalyze the coordinated synthesis of leading and lagging strands, remain stable in the face of dilution provided that the gp44/62 clamp loader, the gp45 sliding clamp, and the gp32 ssDNA-binding protein are present at sufficient levels after dilution. If any of these accessory proteins is omitted from the dilution mixture, uncoordinated DNA synthesis occurs, and/or large Okazaki fragments are formed. Thus, the accessory proteins must be recruited from solution for each round of initiation of lagging-strand synthesis. A modified bacteriophage T7 DNA polymerase (Sequenase) can replace the T4 DNA polymerase for leading-strand synthesis but not for well coordinated lagging-strand synthesis. Although T4 DNA polymerase has been reported to self-associate, gel-exclusion chromatography displays it as a monomer in solution in the absence of DNA. It forms no stable holoenzyme complex in solution with the accessory proteins or with the gp41-gp61 helicase-primase. Instead, template DNA is required for the assembly of the T4 replication complex, which then catalyzes coordinated synthesis of leading and lagging strands in a conditionally coupled manner.

Interacting fidelity defects in the replicative DNA polymerase of bacteriophage RB69.

The DNA polymerases (gp43s) of the related bacteriophages T4 and RB69 are B family (polymerase alpha 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.

Mutation rates among RNA viruses.

The rate of spontaneous mutation is a key parameter in modeling the genetic structure and evolution of populations. The impact of the accumulated load of mutations and the consequences of increasing the mutation rate are important in assessing the genetic health of populations. Mutation frequencies are among the more directly measurable population parameters, although the information needed to convert them into mutation rates is often lacking. A previous analysis of mutation rates in RNA viruses (specifically in riboviruses rather than retroviruses) was constrained by the quality and quantity of available measurements and by the lack of a specific theoretical framework for converting mutation frequencies into mutation rates in this group of organisms. Here, we describe a simple relation between ribovirus mutation frequencies and mutation rates, apply it to the best (albeit far from satisfactory) available data, and observe a central value for the mutation rate per genome per replication of micro(g) approximately 0.76. (The rate per round of cell infection is twice this value or about 1.5.) This value is so large, and ribovirus genomes are so informationally dense, that even a modest increase extinguishes the population.

The distribution of rates of spontaneous mutation over viruses, prokaryotes, and eukaryotes.

Although mutation has chaotic aspects, spontaneous mutation rates assume certain characteristic values when expressed per genome per genome duplication. The rate among lytic RNA viruses is roughly 1, while the rate among retroelements is roughly 0.2. The rate among viral and cellular microbes with DNA chromosomes is close to 0.0034. Mutation rates among higher eukaryotes, estimated from specific-locus studies, vary greatly. Most of this variation can be suppressed if the rates are expressed per cell division instead of per sexual generation, and if the genome size is taken to be only a little larger than the sum of the protein-encoding sequences; then, the mutation rate is roughly 0.01. The reasons for different characteristic mutation rates among different organism groups remain mysterious and pose a substantial challenge to students of evolution.

Lysis and lysis inhibition in bacteriophage T4: rV mutations reside in the holin t gene.

Upon infecting populations of susceptible host cells, T-even bacteriophages maximize their yield by switching from lysis at about 25 to 35 min at 37 degrees C after infection by a single phage particle to long-delayed lysis (lysis inhibition) under conditions of sequential infection occurring when free phages outnumber host cells. The timing of lysis depends upon gene t and upon one or more rapid-lysis (r) genes whose inactivation prevents lysis inhibition. t encodes a holin that mediates the movement of the T4 endolysin though the inner cell membrane to its target, the cell wall. The rI protein has been proposed to sense superinfection. Of the five reasonably well characterized r genes, only two, rI and rV, are clearly obligatory for lysis inhibition. We show here that rV mutations are alleles of t that probably render the t protein unable to respond to the lysis inhibition signal. The tr alleles cluster in the 5' third of t and produce a strong r phenotype, whereas conditional-lethal t alleles produce the classical t phenotype (inability to lyse) and other t alleles produce additional, still poorly understood phenotypes. tr mutations are dominant to t+, a result that suggests specific ways to probe T4 holin function.

Bacteriophage T4 rnh (RNase H) null mutations: effects on spontaneous mutation and epistatic interaction with rII mutations.

The bacteriophage T4 rnh gene encodes T4 RNase H, a relative of a family of flap endonucleases. T4 rnh null mutations reduce burst sizes, increase sensitivity to DNA damage, and increase the frequency of acriflavin resistance (Acr) mutations. Because mutations in the related Saccharomyces cerevisiae RAD27 gene display a remarkable duplication mutator phenotype, we further explored the impact of rnh mutations upon the mutation process. We observed that most Acr mutants in an rnh+ strain contain ac mutations, whereas only roughly half of the Acr mutants detected in an rnhDelta strain bear ac mutations. In contrast to the mutational specificity displayed by most mutators, the DNA alterations of ac mutations arising in rnhDelta and rnh+ backgrounds are indistinguishable. Thus, the increase in Acr mutants in an rnhDelta background is probably not due to a mutator effect. This conclusion is supported by the lack of increase in the frequency of rI mutations in an rnhDelta background. In a screen that detects mutations at both the rI locus and the much larger rII locus, the r frequency was severalfold lower in an rnhDelta background. This decrease was due to the phenotype of rnh rII double mutants, which display an r+ plaque morphology but retain the characteristic inability of rII mutants to grow on lambda lysogens. Finally, we summarize those aspects of T4 forward-mutation systems which are relevant to optimal choices for investigating quantitative and qualitative aspects of the mutation process.

Aspects of the ultraviolet photobiology of some T-even bacteriophages.

Bacteriophage T4 DNA metabolism is largely insulated from that of its host, although some host functions assist in the repair of T4 DNA damage. Environmental factors sometimes affect survival and mutagenesis after ultraviolet (UV) irradiation of T4, and can affect mutagenesis in many organisms. We therefore tested the effect of certain environmental factors and host genetic defects upon spontaneous and UV-induced mutagenesis and survival in T4 and some related T-even phages. Plating at pH 9 enhances UV resistance in T4 by about 14% compared to pH 7. The host cAMP regulatory system affects host survival after UV irradiation but does not affect T4 survival. Thermal rescue, the increasing survival of irradiated T4 with increasing plating temperature, occurs also in phage T6, but only weakly in phages T2 and RB69; this temperature effect is not altered by supplementing infected cells with additional Holliday resolvase (gp49) early in infection. Phage RB69 turns out to have almost 50% greater UV resistance than T4, but has a genome of about the same size; RB69 is UV-mutable but does not produce r mutants, which are easily seen in T2, T4, and T6. Spontaneous mutagenesis in T4 shows no dependence on medium and little dependence on temperature overall, but mutation rates can increase and probably decrease with temperature at specific sites. UV mutagenesis is not affected by incubating irradiated particles under various conditions before plating, in contrast to phage S13.

Rates of spontaneous mutation.

Rates of spontaneous mutation per genome as measured in the laboratory are remarkably similar within broad groups of organisms but differ strikingly among groups. Mutation rates in RNA viruses, whose genomes contain ca. 10(4) bases, are roughly 1 per genome per replication for lytic viruses and roughly 0.1 per genome per replication for retroviruses and a retrotransposon. Mutation rates in microbes with DNA-based chromosomes are close to 1/300 per genome per replication; in this group, therefore, rates per base pair vary inversely and hugely as genome sizes vary from 6 x 10(3) to 4 x 10(7) bases or base pairs. Mutation rates in higher eukaryotes are roughly 0.1-100 per genome per sexual generation but are currently indistinguishable from 1/300 per cell division per effective genome (which excludes the fraction of the genome in which most mutations are neutral). It is now possible to specify some of the evolutionary forces that shape these diverse mutation rates.

Retention of replication fidelity by a DNA polymerase functioning in a distantly related environment.

The primary structures of the replicative DNA polymerases (gp43s) of bacteriophage T4 and its distant phylogenetic relative RB69 are diverged, retaining only 61% identity and 74% similarity. Nevertheless, RB69 gp43 substitutes effectively for T4 gp43 in T4 DNA replication in vivo. We show here that RB69 gp43 replicates T4 genomes in vivo with a fidelity similar to that achieved by T4 gp43. Furthermore, replication by RB69 gp43 in the distantly related environment does not enhance the mutator activities of mutations in T4 genes that encode other components of the multienzyme DNA replicase. We also show that the fidelities of RB69 gp43 and T4 gp43 are both high in vitro and that they are similarly and sharply reduced in vivo by mutations that eliminate the 3'-exonucleolytic proofreading function. We conclude that gp43 interactions with the other replication proteins are probably nonessential for polymerase fidelity.

Rates of spontaneous mutation in bacteriophage T4 are independent of host fidelity determinants.

Bacteriophage T4 encodes most of the genes whose products are required for its DNA metabolism, and host (Escherichia coli) genes can only infrequently complement mutationally inactivated T4 genes. We screened the following host mutator mutations for effects on spontaneous mutation rates in T4: mutT (destruction of aberrant dGTPs), polA, polB and polC (DNA polymerases), dnaQ (exonucleolytic proofreading), mutH, mutS, mutL and uvrD (methyl-directed DNA mismatch repair), mutM and mutY (excision repair of oxygen-damaged DNA), mutA (function unknown), and topB and osmZ (affecting DNA topology). None increased T4 spontaneous mutation rates within a resolving power of about twofold (nor did optA, which is not a mutator but overexpresses a host dGTPase). Previous screens in T4 have revealed strong mutator mutations only in the gene encoding the viral DNA polymerase and proofreading 3'-exonuclease, plus weak mutators in several polymerase accessory proteins or determinants of dNTP pool sizes. T4 maintains a spontaneous mutation rate per base pair about 30-fold greater than that of its host. Thus, the joint high fidelity of insertion by T4 DNA polymerase and proofreading by its associated 3'-exonuclease appear to determine the T4 spontaneous mutation rate, whereas the host requires numerous additional systems to achieve high replication fidelity.

Looking backward on a century of mutation research.

Our understanding of the mutation process and how it impacts target populations has deepened steadily during the hundred years since 1919. Recent advances in engineering genetically determined social traits points towards the logical culmination of this knowledge.

Rates of spontaneous mutation among RNA viruses.

Simple methods are presented to estimate rates of spontaneous mutation from mutant frequencies and population parameters in RNA viruses. Published mutant frequencies yield a wide range of mutation rates per genome per replication, mainly because mutational targets have usually been small and, thus, poor samples of the mutability of the average base. Nevertheless, there is a clear central tendency for lytic RNA viruses (bacteriophage Q beta, poliomyelitis, vesicular stomatitis, and influenza A) to display rates of spontaneous mutation of approximately 1 per genome per replication. This rate is some 300-fold higher than previously reported for DNA-based microbes. Lytic RNA viruses thus mutate at a rate close to the maximum value compatible with viability. Retroviruses (spleen necrosis, murine leukemia, Rous sarcoma), however, mutate at an average rate about an order of magnitude lower than lytic RNA viruses.

General antimutators are improbable.

Antimutator mutations reduce spontaneous mutation rates, at least at some sites and along some pathways. Antimutators have been found in several microbial systems since their initial discovery in bacteriophage T4, where they occur mainly among mutations of gene 43 (which encodes the viral DNA polymerase). The phage T4 antimutators are highly specific, often strongly reducing mutations rates but only along specific pathways, usually A.T-->G.C. They may fail to affect other pathways, such as G.C-->A.T, and may even accelerate mutation at yet other pathways, such as transversions (R.Y-->Y.R). Both enzymatic and evolutionary considerations suggest that it should be difficult to isolate strong, general antimutator mutations, that is, mutations that substantially lower the total spontaneous mutation rate over the entire genome without producing strongly deleterious side effects. This notion has been tested by measuring mutation rates over a target comprising several kilobases in a set of phage T4 antimutators. In each case, this rate was indistinguishable from or greater than the wild-type rate. A survey of reports describing antimutators in other microbes reveals that none are yet demonstrated to be general antimutators.

Duplication-targeted DNA methylation and mutagenesis in the evolution of eukaryotic chromosomes.

Mammalian genomes are threatened with gene inactivation and chromosomal scrambling by recombination between repeated sequences such as mobile genetic elements and pseudogenes. We present and test a model for a defensive strategy based on the methylation and subsequent mutation of CpG dinucleotides in those DNA duplications that create uninterrupted homologous sequences longer than about 0.3 kilobases. The model helps to explain both the diversity of CpG frequencies in different genes and the persistence of gene fragmentation into exons and introns.