RESUMO
Selection of spontaneous, loss-of-function mutations at two chromosomal loci (pyrF and pyrE) enabled the first molecular-level analysis of replication fidelity in the extremely thermophilic bacterium Thermus thermophilus. Two different methods yielded similar mutation rates, and mutational spectra determined by sequencing of independent mutants revealed a variety of replication errors distributed throughout the target genes. The genomic mutation rate estimated from these targets, 0.00097 +/- 0.00052 per replication, was lower than corresponding estimates from mesophilic microorganisms, primarily because of a low rate of base substitution. However, both the rate and spectrum of spontaneous mutations in T. thermophilus resembled those of the thermoacidophilic archaeon Sulfolobus acidocaldarius, despite important molecular differences between these two thermophiles and their genomes.
Assuntos
Regulação Bacteriana da Expressão Gênica , Mutação , Thermus thermophilus/genética , Sequência de Bases , Códon , Análise Mutacional de DNA , Regulação da Expressão Gênica em Archaea , Genoma Arqueal , Genoma Bacteriano , Modelos Genéticos , Dados de Sequência Molecular , Sulfolobus acidocaldarius/genéticaRESUMO
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 approximately 2 x 10(-8)/cell division in fluctuation and null-fraction tests but approximately 6 x 10(-8)/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.
Assuntos
Genes Arqueais/genética , Haloferax volcanii/genética , Mutação/genética , Haloferax volcanii/enzimologia , Orotato Fosforribosiltransferase/genética , Sequências Repetitivas de Ácido Nucleico/genéticaRESUMO
Replication repair mediates error-free bypass of DNA damage in a series of steps that include regression of the replication fork, primer-terminus switching to use the other daughter strand as an undamaged template, primer extension, primer switching back to its cognate template with the primer terminus now having bypassed the damage, and fork rearrangement to a normal configuration. By both genetic and biochemical criteria, bacteriophage T4 catalyzes replication repair with two alternative sets of proteins, one including the gp32 SSB and the gp41 DNA helicase and the other including the UvsX recombinase. In each pathway, synthesis is conducted by the gp43 DNA polymerase. Here we show that defects in gp32, gp41 or UvsX that impair replication repair also increase mutation rates generally, but especially for templated mutations. Such templated mutations are associated with palindromic or direct repeats that are either perfect or imperfect. Models of templated mutagenesis require that the primer terminus switches to an ectopic template, but one that yields mutations instead of error-free bypass. We suggest that the proteins that conduct replication repair normally direct a blocked primer strand specifically to the other daughter strand with considerable accuracy, but that strand switching becomes promiscuous when these proteins are mutationally impaired, thus promoting templated mutations.
Assuntos
Reparo do DNA , Replicação do DNA , Mutação , Bacteriófago T4/genética , Bacteriófago T4/metabolismo , Sequência de Bases , Dano ao DNA , DNA Viral/genética , DNA Viral/metabolismo , Escherichia coli/virologia , Genes Virais , Modelos Genéticos , Dados de Sequência MolecularRESUMO
Numerous studies of the impact of accessory proteins upon the fidelity of DNA synthesis have provided a complex and sometimes discordant picture. We previously described such an analysis conducted in vitro using various bacteriophage RB69 gp43 mutator DNA polymerases with or without the accessory proteins gp32 (which binds single-stranded DNA) plus gp45/44/62 (processivity clamp and its loaders). Mutations were scored at many sites in the lacZalpha mutation reporter sequence. Unexpectedly, the accessory proteins sometimes decreased and sometimes increased fidelity at a handful of specific sites. Here, we enlarge our analysis with one particular mutator polymerase compromised in both insertion accuracy and proofreading and also extend the analysis to reactions supplemented only with gp32 or only with gp45/44/62. An overall 1.56-fold increase in mutation frequencies was produced by adding single or multiple accessory proteins and was driven mainly by increased T(template)*G(primer) mispairs. Evidence was found for many additional sites where the accessory proteins influence fidelity, indicating the generality of the effect. Thus, accessory proteins contribute to the site-specific variability in mutation rates characteristically seen in mutational spectra.
Assuntos
Bacteriófago T4/química , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/fisiologia , Transativadores/fisiologia , Proteínas Virais/química , Proteínas Virais/fisiologia , Bacteriófagos/metabolismo , Sítios de Ligação , Análise Mutacional de DNA , Replicação do DNA , DNA Viral/química , Proteínas de Ligação a DNA/química , Genes Reporter , Óperon Lac , Substâncias Macromoleculares/química , Modelos Genéticos , Método de Monte Carlo , Mutação , Ligação Proteica , Transativadores/químicaRESUMO
Bacteriophage RB69 encodes a replicative B-family DNA polymerase (RB69 gp43) with an associated proofreading 3' exonuclease. Crystal structures have been determined for this enzyme with and without DNA substrates. We previously described the mutation rates and kinds of mutations produced in vivo by the wild-type (Pol(+) Exo(+)) enzyme, an exonuclease-deficient mutator variant (Pol(+) Exo(-)), mutator variants with substitutions at Tyr(567) in the polymerase active site (Pol(M) Exo(+)), and the double mutator Pol(M) Exo(-). Comparing the mutational spectra of the Pol(+) Exo(-) and Pol(+) Exo(+) enzymes revealed the patterns and efficiencies of proofreading, while Tyr(567) was identified as an important determinant of base-selection fidelity. Here, we sought to determine how well the fidelities of the same enzymes are reflected in vitro. Compared to their behavior in vivo, the three mutator polymerases exhibited modestly higher mutation rates in vitro and their mutational predilections were also somewhat different. Although the RB69 gp43 accessory proteins exerted little or no effect on total mutation rates in vitro, they strongly affected mutation rates at many specific sites, increasing some rates and decreasing others.
Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Mutação , Proteínas Virais/metabolismo , Bacteriófago M13/genética , Sequência de Bases , Técnicas In Vitro , Dados de Sequência MolecularRESUMO
Phage RB69 B-family DNA polymerase is responsible for the overall high fidelity of RB69 DNA synthesis. Fidelity is compromised when conserved Tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. The Y567A mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. Ser565 is a nearby conserved residue that also contributes to the binding pocket, but a S565G replacement has only a small impact on DNA replication fidelity. When Y567A and S565G replacements were combined, mutator activity was strongly decreased compared to that with Y567A replacement alone. Analyses conducted both in vivo and in vitro revealed that, compared to Y567A replacement alone, the double mutant mainly reduced base substitution mutations and, to a lesser extent, frameshift mutations. The decrease in mutation rates was not due to increased exonuclease activity. Based on measurements of DNA binding affinity, mismatch insertion, and mismatch extension, we propose that the recovered fidelity of the double mutant may result, in part, from an increased dissociation of the enzyme from DNA, followed by the binding of the same or another polymerase molecule in either exonuclease mode or polymerase mode. An additional antimutagenic factor may be a structural alteration in the polymerase binding pocket described in this article.