Rodrigo S. Galhardo

Mutation as a Stress Response and the Regulation of Evolvability

ABSTRACT Our concept of a stable genome is evolving to one in which genomes are plastic and responsive to environmental changes. Growing evidence shows that a variety of environmental stresses induce genomic instability in bacteria, yeast, and human cancer cells, generating occasional fitter mutants and potentially accelerating adaptive evolution. The emerging molecular mechanisms of stress-induced mutagenesis vary but share telling common components that underscore two common themes. The first is the regulation of mutagenesis in time by cellular stress responses, which promote random mutations specifically when cells are poorly adapted to their environments, i.e., when they are stressed. A second theme is the possible restriction of random mutagenesis in genomic space, achieved via coupling of mutation-generating machinery to local events such as DNA-break repair or transcription. Such localization may minimize accumulation of deleterious mutations in the genomes of rare fitter mutants, and promote local concerted evolution. Although mutagenesis induced by stresses other than direct damage to DNA was previously controversial, evidence for the existence of various stress-induced mutagenesis programs is now overwhelming and widespread. Such mechanisms probably fuel evolution of microbial pathogenesis and antibiotic-resistance, and tumor progression and chemotherapy resistance, all of which occur under stress, driven by mutations. The emerging commonalities in stress-induced-mutation mechanisms provide hope for new therapeutic interventions for all of these processes.

DinB upregulation is the sole role of the SOS response in stress-induced mutagenesis in Escherichia coli

Stress-induced mutagenesis is a collection of mechanisms observed in bacterial, yeast, and human cells in which adverse conditions provoke mutagenesis, often under the control of stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e., are stressed. It is therefore important to understand how stress responses increase mutagenesis. In the Escherichia coli Lac assay, stress-induced point mutagenesis requires induction of at least two stress responses: the RpoS-controlled general/starvation stress response and the SOS DNA-damage response, both of which upregulate DinB error-prone DNA polymerase, among other genes required for Lac mutagenesis. We show that upregulation of DinB is the only aspect of the SOS response needed for stress-induced mutagenesis. We constructed two dinB(oc) (operator-constitutive) mutants. Both produce SOS-induced levels of DinB constitutively. We find that both dinB(oc) alleles fully suppress the phenotype of constitutively SOS-“off” lexA(Ind−) mutant cells, restoring normal levels of stress-induced mutagenesis. Thus, dinB is the only SOS gene required at induced levels for stress-induced point mutagenesis. Furthermore, although spontaneous SOS induction has been observed to occur in only a small fraction of cells, upregulation of dinB by the dinB(oc) alleles in all cells does not promote a further increase in mutagenesis, implying that SOS induction of DinB, although necessary, is insufficient to differentiate cells into a hypermutable condition.

An SOS-regulated operon involved in damage-inducible mutagenesis in Caulobacter crescentus

DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the catalytic subunit of Pol III, is damage inducible in a recA-dependent manner, and is responsible for most ultraviolet (UV) and mitomycin C-induced mutations in C.crescentus. The results demonstrate that the three genes are required for the error-prone processing of DNA lesions. The two hypothetical genes were named imuA and imuB, after inducible mutagenesis. ImuB is similar to proteins of the Y-family of polymerases, and possibly cooperates with DnaE2 in lesion bypass. The mutations arising as a consequence of the activity of the imuAB dnaE2 operon are rather unusual for UV irradiation, including G:C to C:G transversions.

DNA damage by singlet oxygen and cellular protective mechanisms

Reactive oxygen species, as singlet oxygen ((1)O(2)) and hydrogen peroxide, are continuously generated by aerobic organisms, and react actively with biomolecules. At excessive amounts, (1)O(2) induces oxidative stress and shows carcinogenic and toxic effects due to oxidation of lipids, proteins and nucleic acids. Singlet oxygen is able to react with DNA molecule and may induce G to T transversions due to 8-oxodG generation. The nucleotide excision repair, base excision repair and mismatch repair have been implicated in the correction of DNA lesions induced by (1)O(2) both in prokaryotic and in eukaryotic cells. (1)O(2) is also able to induce the expression of genes involved with the cellular responses to oxidative stress, such as NF-κB, c-fos and c-jun, and genes involved with tissue damage and inflammation, as ICAM-1, interleukins 1 and 6. The studies outlined in this review reinforce the idea that (1)O(2) is one of the more dangerous reactive oxygen species to the cells, and deserves our attention.

The σE stress response is required for stress-induced mutation and amplification in Escherichia coli

Pathways of mutagenesis are induced in microbes under adverse conditions controlled by stress responses. Control of mutagenesis by stress responses may accelerate evolution specifically when cells are maladapted to their environments, i.e. are stressed. Stress‐induced mutagenesis in the Escherichia coli Lac assay occurs either by ‘point’ mutation or gene amplification. Point mutagenesis is associated with DNA double‐strand‐break (DSB) repair and requires DinB error‐prone DNA polymerase and the SOS DNA‐damage‐ and RpoS general‐stress responses. We report that the RpoE envelope‐protein‐stress response is also required. In a screen for mutagenesis‐defective mutants, we isolated a transposon insertion in the rpoE P2 promoter. The insertion prevents rpoE induction during stress, but leaves constitutive expression intact, and allows cell viability. rpoE insertion and suppressed null mutants display reduced point mutagenesis and maintenance of amplified DNA. Furthermore, σE acts independently of stress responses previously implicated: SOS/DinB and RpoS, and of σ32, which was postulated to affect mutagenesis. I‐SceI‐induced DSBs alleviated much of the rpoE phenotype, implying that σE promoted DSB formation. Thus, a third stress response and stress input regulate DSB‐repair‐associated stress‐induced mutagenesis. This provides the first report of mutagenesis promoted by σE, and implies that extracytoplasmic stressors may affect genome integrity and, potentially, the ability to evolve.

Characterization of the SOS Regulon of Caulobacter crescentus

The SOS regulon is a paradigm of bacterial responses to DNA damage. A wide variety of bacterial species possess homologs of lexA and recA, the central players in the regulation of the SOS circuit. Nevertheless, the genes actually regulated by the SOS have been determined only experimentally in a few bacterial species. In this work, we describe 37 genes regulated in a LexA-dependent manner in the alphaproteobacterium Caulobacter crescentus. In agreement with previous results, we have found that the direct repeat GTTCN7GTTC is the SOS operator of C. crescentus, which was confirmed by site-directed mutagenesis studies of the imuA promoter. Several potential promoter regions containing the SOS operator were identified in the genome, and the expression of the corresponding genes was analyzed for both the wild type and the lexA strain, demonstrating that the vast majority of these genes are indeed SOS regulated. Interestingly, many of these genes encode proteins with unknown functions, revealing the potential of this approach for the discovery of novel genes involved in cellular responses to DNA damage in prokaryotes, and illustrating the diversity of SOS-regulated genes among different bacterial species.

Structure of the Thiazole Biosynthetic Enzyme THI1 from Arabidopsis thaliana

Thiamin pyrophosphate is an essential coenzyme in all organisms that depend on fermentation, respiration or photosynthesis to produce ATP. It is synthesized through two independent biosynthetic routes: one for the synthesis of 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate (pyrimidine moiety) and another for the synthesis of 4-methyl-5-(β-hydroxyethyl) thiazole phosphate (thiazole moiety). Herein, we will describe the three-dimensional structure of THI1 protein from Arabidopsis thaliana determined by single wavelength anomalous diffraction to 1.6Å resolution. The protein was produced using heterologous expression in bacteria, unexpectedly bound to 2-carboxylate-4-methyl-5-β-(ethyl adenosine 5—diphosphate) thiazole, a potential intermediate of the thiazole biosynthesis in Eukaryotes. THI1 has a topology similar to dinucleotide binding domains and although details concerning its function are unknown, this work provides new clues about the thiazole biosynthesis in Eukaryotes.

Repair of DNA Lesions Induced by Hydrogen Peroxide in the Presence of Iron Chelators in Escherichia coli: Participation of Endonuclease IV and Fpg

In Escherichia coli, the repair of lethal DNA damage induced by H(2)O(2) requires exonuclease III, the xthA gene product. Here, we report that both endonuclease IV (the nfo gene product) and exonuclease III can mediate the repair of lesions induced by H(2)O(2) under low-iron conditions. Neither the xthA nor the nfo mutants was sensitive to H(2)O(2) in the presence of iron chelators, while the xthA nfo double mutant was significantly sensitive to this treatment, suggesting that both exonuclease III and endonuclease IV can mediate the repair of DNA lesions formed under such conditions. Sedimentation studies in alkaline sucrose gradients also demonstrated that both xthA and nfo mutants, but not the xthA nfo double mutant, can carry out complete repair of DNA strand breaks and alkali-labile bonds generated by H(2)O(2) under low-iron conditions. We also found indications that the formation of substrates for exonuclease III and endonuclease IV is mediated by the Fpg DNA glycosylase, as suggested by experiments in which the fpg mutation increased the level of cell survival, as well as repair of DNA strand breaks, in an AP endonuclease-null background.

DNA repair-related genes in sugarcane expressed sequence tags (ESTs)

There is much interest in the identification and characterization of genes involved in DNA repair because of their importance in the maintenance of the genome integrity. The high level of conservation of DNA repair genes means that these genetic elements may be used in phylogenetic studies as a source of information on the genetic origin and evolution of species. The mechanisms by which damaged DNA is repaired are well understood in bacteria, yeast and mammals, but much remains to be learned as regards plants. We identified genes involved in DNA repair mechanisms in sugarcane using a similarity search of the Brazilian Sugarcane Expressed Sequence Tag (SUCEST) database against known sequences deposited in other public databases (National Center of Biotechnology Information (NCBI) database and the Munich Information Center for Protein Sequences (MIPS) Arabidopsis thaliana database). This search revealed that most of the various proteins involved in DNA repair in sugarcane are similar to those found in other eukaryotes. However, we also identified certain intriguing features found only in plants, probably due to the independent evolution of this kingdom. The DNA repair mechanisms investigated include photoreactivation, base excision repair, nucleotide excision repair, mismatch repair, non-homologous end joining, homologous recombination repair and DNA lesion tolerance. We report the main differences found in the DNA repair machinery in plant cells as compared to other organisms. These differences point to potentially different strategies plants employ to deal with DNA damage, that deserve further investigation.

Functional characterization of two SOS-regulated genes involved in mitomycin C resistance in Caulobacter crescentus

The SOS response is a universal bacterial regulon involved in the cellular response to DNA damage and other forms of stress. In Caulobacter crescentus, previous work has identified a plethora of genes that are part of the SOS regulon, but the biological roles of several of them remain to be determined. In this study, we report that two genes, hereafter named mmcA and mmcB, are involved in the defense against DNA damage caused by mitomycin C (MMC), but not against lesions induced by other common DNA damaging agents, such as UVC light, methyl methanesulfonate (MMS) and hydrogen peroxide. mmcA is a conserved gene that encodes a member of the glyoxalases/dioxygenases protein family, and acts independently of known DNA repair pathways. On the other hand, epistasis analysis showed that mmcB acts in the same pathway as imuC (dnaE2), and is required specifically for MMC-induced mutagenesis, but not for that induced by UV light, suggesting a role for MmcB in translesion synthesis-dependent repair of MMC damage. We show that the lack of MMC-induced mutability in the mmcB strain is not caused by lack of proper SOS induction of the imuABC operon, involved in translesion synthesis (TLS) in C. crescentus. Based on this data and on structural analysis of a close homolog, we propose that MmcB is an endonuclease which creates substrates for ImuABC-mediated TLS patches.