Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning.

Although base editors are widely used to install targeted point mutations, the factors that determine base editing outcomes are not well understood. We characterized sequence-activity relationships of 11 cytosine and adenine base editors (CBEs and ABEs) on 38,538 genomically integrated targets in mammalian cells and used the resulting outcomes to train BE-Hive, a machine learning model that accurately predicts base editing genotypic outcomes (R ≈ 0.9) and efficiency (R ≈ 0.7). We corrected 3,388 disease-associated SNVs with ≥90% precision, including 675 alleles with bystander nucleotides that BE-Hive correctly predicted would not be edited. We discovered determinants of previously unpredictable C-to-G, or C-to-A editing and used these discoveries to correct coding sequences of 174 pathogenic transversion SNVs with ≥90% precision. Finally, we used insights from BE-Hive to engineer novel CBE variants that modulate editing outcomes. These discoveries illuminate base editing, enable editing at previously intractable targets, and provide new base editors with improved editing capabilities.

Molnupiravir increases SARS-CoV-2 genome diversity and complexity: A case-control cohort study.

Molnupiravir, an oral direct-acting antiviral effective in vitro against SARS-CoV-2, has been largely employed during the COVID-19 pandemic, since December 2021. After marketing and widespread usage, a progressive increase in SARS-CoV-2 lineages characterized by a higher transition/transversion ratio, a characteristic signature of molnupiravir action, appeared in the Global Initiative on Sharing All Influenza Data (GISAID) and International Nucleotide Sequence Database Collaboration (INSDC) databases. Here, we assessed the drug effects by SARS-CoV-2 whole-genome sequencing on 38 molnupiravir-treated persistently positive COVID-19 outpatients tested before and after treatment. Seventeen tixagevimab/cilgavimab-treated outpatients served as controls. Mutational analyses confirmed that SARS-CoV-2 exhibits an increased transition/transversion ratio seven days after initiation of molnupiravir. Moreover we observed an increased G->A ratio compared to controls, which was not related to apolipoprotein B mRNAediting enzyme, catalytic polypeptide-like (APOBEC) activity. In addition, we demonstrated for the first time an increased diversity and complexity of the viral quasispecies.

Detection of Transversions and Transitions in HBG2 Cis-Elements Associated with Sickle Cell Allele in Ghanaians.

Short tandem repeats located 5' prime to the ß-globin gene, have been observed to be in linkage disequilibrium with the HbS allele, and thought to affect the severity of sickle cell disease. Here, we report on new mutants within the HBG2 region that may impact sickle cell disease. To determine the cis-acting elements microsatellites, indels and single nucleotide polymorphisms (SNPs), within the HBG2 region by sequencing, in subjects with sickle cell disease. The case-control study was located at the Center for Clinical Genetics, Sickle cell unit, Korle-Bu Teaching Hospital. A questionnaire was used for demographic data and clinical information. Hematological profile (red blood cell, white blood cell, platelet, hemoglobin and mean corpuscular volume) were assessed in 83 subjects. A set of 45 samples comprising amplified DNA on the HBG2 gene from HbSS (22), HbSC (17) and 6 controls (HbAA) were sequenced. Differences in the microsatellite region between sickle cell disease (SCD) (HbSS and HbSC) genotypes and control subjects were identified by counting and assessed by Chi-square analysis. Red blood cells, hematocrit, platelets, white blood cells and hemoglobin indices differed in genotypic groups. HbSS subjects were affirmed to have severer hemolytic anemia than HbSC subjects. Two indels (T1824 and C905) were seen in both SS and SC genotypes. Two peculiar SNPs: G:T1860 (transition) and A:G1872 transversions were found within the HBG2 gene that were significantly associated with the HbSS genotype (Fisher's exact test, p = 0.006) and HbS allele respectively (Fisher's exact test, p = 0.006). Cis-acting elements in HbSS and HbSC were different and may contribute to the phenotype seen in the disease state.

Translation of Mutant Repetitive Genomic Sequences in Hirsutella sinensis and Changes in the Secondary Structures and Functional Specifications of the Encoded Proteins.

Multiple repetitive sequences of authentic genes commonly exist in fungal genomes. AT-biased genotypes of Ophiocordyceps sinensis have been hypothesized as repetitive pseudogenes in the genome of Hirsutella sinensis (GC-biased Genotype #1 of O. sinensis) and are generated through repeat-induced point mutation (RIP), which is charactered by cytosine-to-thymine and guanine-to-adenine transitions, concurrent epigenetic methylation, and dysfunctionality. This multilocus study examined repetitive sequences in the H. sinensis genome and transcriptome using a bioinformatic approach and revealed that 8.2% of the authentic genes had repetitive copies, including various allelic insertions/deletions, transversions, and transitions. The transcripts for the repetitive sequences, regardless of the decreases, increases, or bidirectional changes in the AT content, were identified in the H. sinensis transcriptome, resulting in changes in the secondary protein structure and functional specification. Multiple repetitive internal transcribed spacer (ITS) copies containing multiple insertion/deletion and transversion alleles in the genome of H. sinensis were GC-biased and were theoretically not generated through RIP mutagenesis. The repetitive ITS copies were genetically and phylogenetically distinct from the AT-biased O. sinensis genotypes that possess multiple transition alleles. The sequences of Genotypes #2-17 of O. sinensis, both GC- and AT-biased, were absent from the H. sinensis genome, belong to the interindividual fungi, and differentially occur in different compartments of the natural Cordyceps sinensis insect-fungi complex, which contains >90 fungal species from >37 genera. Metatranscriptomic analyses of natural C. sinensis revealed the transcriptional silencing of 5.8S genes in all C. sinensis-colonizing fungi in natural settings, including H. sinensis and other genotypes of O. sinensis. Thus, AT-biased genotypes of O. sinensis might have evolved through advanced evolutionary mechanisms, not through RIP mutagenesis, in parallel with GC-biased Genotype #1 of H. sinensis from a common genetic ancestor over the long course of evolution.

Base Editing Landscape Extends to Perform Transversion Mutation.

Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.

Are Nonsynonymous Transversions Generally More Deleterious than Nonsynonymous Transitions?

It has been suggested that, due to the structure of the genetic code, nonsynonymous transitions are less likely than transversions to cause radical changes in amino acid physicochemical properties so are on average less deleterious. This view was supported by some but not all mutagenesis experiments. Because laboratory measures of fitness effects have limited sensitivities and relative frequencies of different mutations in mutagenesis studies may not match those in nature, we here revisit this issue using comparative genomics. We extend the standard codon model of sequence evolution by adding the parameter η that quantifies the ratio of the fixation probability of transitional nonsynonymous mutations to that of transversional nonsynonymous mutations. We then estimate η from the concatenated alignment of all protein-coding DNA sequences of two closely related genomes. Surprisingly, η ranges from 0.13 to 2.0 across 90 species pairs sampled from the tree of life, with 51 incidences of η < 1 and 30 incidences of η >1 that are statistically significant. Hence, whether nonsynonymous transversions are overall more deleterious than nonsynonymous transitions is species-dependent. Because the corresponding groups of amino acid replacements differ between nonsynonymous transitions and transversions, η is influenced by the relative exchangeabilities of amino acid pairs. Indeed, an extensive search reveals that the large variation in η is primarily explainable by the recently reported among-species disparity in amino acid exchangeabilities. These findings demonstrate that genome-wide nucleotide substitution patterns in coding sequences have species-specific features and are more variable among evolutionary lineages than are currently thought.

Stem Region of tRNA Genes Favors Transition Substitution Towards Keto Bases in Bacteria.

Transversion and transition mutations have variable effects on the stability of RNA secondary structure considering that the former destabilizes the double helix geometry to a greater extent by introducing purine:purine (R:R) or pyrimidine:pyrimidine (Y:Y) base pairs. Therefore, transversion frequency is likely to be lower than that of transition in the secondary structure regions of RNA genes. Here, we performed an analysis of transition and transversion frequencies in tRNA genes defined well with secondary structure and compared with the intergenic regions in five bacterial species namely Escherichia coli, Klebsiella pneumoniae, Salmonella enterica, Staphylococcus aureus and Streptococcus pneumoniae using a large genome sequence data set. In general, the transversion frequency was observed to be lower than that of transition in both tRNA genes and intergenic regions. The transition to transversion ratio was observed to be greater in tRNA genes than that in the intergenic regions in all the five bacteria that we studied. Interestingly, the intraspecies base substitution analysis in tRNA genes revealed that non-compensatory substitutions were more frequent than compensatory substitutions in the stem region. Further, transition to transversion ratio in the loop region was observed to be significantly lesser than that among the non-compensatory substitutions in the stem region. This indicated that the transversion is more deleterious than transition in the stem regions. In addition, substitutions from amino bases (A/C) to keto bases (G/T) were also observed to be more than the reverse substitutions in the stem region. Substitution from amino bases to keto bases are likely to facilitate the stable G:U pairing unlike the reverse substitution that facilitates the unstable A:C pairing in the stem region of tRNA. This work provides additional support that the secondary structure of tRNA molecule is what drives the different substitutions in its gene sequence.

Differential properties of KRAS transversion and transition mutations in non-small cell lung cancer: associations with environmental factors and clinical outcomes.

BACKGROUND: KRAS-mutated non-small cell lung cancer (NSCLC) accounts for 23-35% and 13-20% of all NSCLCs in white patients and East Asians, respectively, and is therefore regarded as a major therapeutic target. However, its epidemiology and clinical characteristics have not been fully elucidated because of its wide variety of mutational subtypes. Here, we focused on two distinct base substitution types: transversion mutations and transition mutations, as well as their association with environmental factors and clinical outcome. METHODS: Dataset from the Japan Molecular Epidemiology Study, which is a prospective, multicenter, and molecular study epidemiology cohort study involving 957 NSCLC patients who underwent surgery, was used for this study. Questionnaire-based detailed information on clinical background and lifestyles was also used to assess their association with mutational subtypes. Somatic mutations in 72 cancer-related genes were analyzed by next-generation sequencing, and KRAS mutations were classified into three categories: transversions (G > C or G > T; G12A, G12C, G12R, G12V), transitions (G > A; G12D, G12S, G13D), and wild-type (WT). Clinical correlations between these subtypes have been investigated, and recurrence-free survival (RFS) and overall survival (OS) were evaluated. RESULTS: Of the 957 patients, KRAS mutations were detected in 80 (8.4%). Of these, 61 were transversions and 19 were transitions mutations. Both pack-years of smoking and smoking duration had significant positive correlation with the occurrence of transversion mutations (p = 0.03 and < 0.01, respectively). Notably, transitions showed an inverse correlation with vegetable intake (p = 0.01). Patients with KRAS transitions had the shortest RFS and OS compared to KRAS transversions and WT. Multivariate analysis revealed that KRAS transitions, along with age and stage, were significant predictors of shorter RFS and OS (HR 2.15, p = 0.01; and HR 2.84, p < 0.01, respectively). CONCLUSIONS: Smoking exposure positively correlated with transversions occurrence in a dose-dependent manner. However, vegetable intake negatively correlated with transitions. Overall, KRAS transition mutations are significantly poor prognostic factors among resected NSCLC patients.

Selection signatures in melanocortin-1 receptor gene of turkeys (Meleagris gallopavo) raised in hot humid tropics.

Feather colours are used by avian species for defense, adaptation and signaling. Melanocortin-1 receptor (MC1R) gene is one of the genes responsible for feather colour. This study identified selection signatures in MC1R gene of Nigerian indigenous turkeys (NIT) using British United turkeys (BUT) as control breed to investigate the evolutionary processes that have shaped NIT with various feather colours. Complete MC1R gene of 146 NIT (76 males and 70 females) and 32 BUT (18 males and 14 females) were sequenced. Transition/transversion and codon usage biases were predicted using MEGA v6 software. The selective force acting on the gene was predicted using HyPhy software. The FST values were estimated using Arlequin v3.5. The highest transition/transversion bias was predicted for white BUT (1.00) while the lowest was predicted for black NIT (0.50). Negative dN-dS values, indicative of purifying selection, were observed in MC1R gene of all the turkeys. The highest pairwise FST was observed between the MC1R gene of white BUT and black NIT while the least was observed between lavender NIT and white NIT. No recombination event was observed in black NIT and white BUT. The relative synonymous codon usage was the same among different colours for some codons. Presence of purifying selection in MC1R gene of all the turkeys with different feather colours confirms that the gene plays role in many biological processes such as feather colouration, behaviour, pain perception, immunity, growth and adaptation. The results also suggested that the genetic mechanisms generating different feather colours in turkeys are conserved.

Spontaneous and photosensitization-induced mutations in primary mouse cells transitioning through senescence and immortalization.

To investigate the role of oxidative stress-induced DNA damage and mutagenesis in cellular senescence and immortalization, here we profiled spontaneous and methylene blue plus light-induced mutations in the cII gene from λ phage in transgenic mouse embryonic fibroblasts during the transition from primary culture through senescence and immortalization. Consistent with detection of characteristic oxidized guanine lesions (8-oxodG) in the treated cells, we observed significantly increased relative cII mutant frequency in the treated pre-senescent cells which was augmented in their immortalized counterparts. The predominant mutation type in the treated pre-senescent cells was G:C→T:A transversion, whose frequency was intensified in the treated immortalized cells. Conversely, the prevailing mutation type in the treated immortalized cells was A:T→C:G transversion, with a unique sequence-context specificity, i.e. flanking purines at the 5' end of the mutated nucleotide. This mutation type was also enriched in the treated pre-senescent cells, although to a lower extent. The signature mutation of G:C→T:A transversions in the treated cells accorded with the well-established translesion synthesis bypass caused by 8-oxodG, and the hallmark A:T→C:G transversions conformed to the known replication errors because of oxidized guanine nucleosides (8-OHdGTPs). The distinctive features of photosensitization-induced mutagenesis in the immortalized cells, which were present at attenuated levels, in spontaneously immortalized cells provide insights into the role of oxidative stress in senescence bypass and immortalization. Our results have important implications for cancer biology because oxidized purines in the nucleoside pool can significantly contribute to genetic instability in DNA mismatch repair-defective human tumors.

Trends of mutation accumulation across global SARS-CoV-2 genomes: Implications for the evolution of the novel coronavirus.

To understand SARS-CoV-2 microevolution, this study explored the genome-wide frequency, gene-wise distribution, and molecular nature of all point-mutations detected across its 71,703 RNA-genomes deposited in GISAID till 21 August 2020. Globally, nsp1/nsp2 and orf7a/orf3a were the most mutation-ridden non-structural and structural genes respectively. Phylogeny of 4618 spatiotemporally-representative genomes revealed that entities belonging to the early lineages are mostly spread over Asian countries, including India, whereas the recently-derived lineages are more globally distributed. Of the total 20,163 instances of polymorphism detected across global genomes, 12,594 and 7569 involved transitions and transversions, predominated by cytidine-to-uridine and guanosine-to-uridine conversions, respectively. Positive selection of nonsynonymous mutations (dN/dS >1) in most of the structural, but not the non-structural, genes indicated that SARS-CoV-2 has already harmonized its replication/transcription machineries with the host metabolism, while it is still redefining virulence/transmissibility strategies at the molecular level. Mechanistic bases and evolutionary/pathogenicity-related implications are discussed for the predominant mutation-types.

Genome-Wide DNA Alterations in X-Irradiated Human Gingiva Fibroblasts.

While ionizing radiation (IR) is a powerful tool in medical diagnostics, nuclear medicine, and radiology, it also is a serious threat to the integrity of genetic material. Mutagenic effects of IR to the human genome have long been the subject of research, yet still comparatively little is known about the genome-wide effects of IR exposure on the DNA-sequence level. In this study, we employed high throughput sequencing technologies to investigate IR-induced DNA alterations in human gingiva fibroblasts (HGF) that were acutely exposed to 0.5, 2, and 10 Gy of 240 kV X-radiation followed by repair times of 16 h or 7 days before whole-genome sequencing (WGS). Our analysis of the obtained WGS datasets revealed patterns of IR-induced variant (SNV and InDel) accumulation across the genome, within chromosomes as well as around the borders of topologically associating domains (TADs). Chromosome 19 consistently accumulated the highest SNVs and InDels events. Translocations showed variable patterns but with recurrent chromosomes of origin (e.g., Chr7 and Chr16). IR-induced InDels showed a relative increase in number relative to SNVs and a characteristic signature with respect to the frequency of triplet deletions in areas without repetitive or microhomology features. Overall experimental conditions and datasets the majority of SNVs per genome had no or little predicted functional impact with a maximum of 62, showing damaging potential. A dose-dependent effect of IR was surprisingly not apparent. We also observed a significant reduction in transition/transversion (Ti/Tv) ratios for IR-dependent SNVs, which could point to a contribution of the mismatch repair (MMR) system that strongly favors the repair of transitions over transversions, to the IR-induced DNA-damage response in human cells. Taken together, our results show the presence of distinguishable characteristic patterns of IR-induced DNA-alterations on a genome-wide level and implicate DNA-repair mechanisms in the formation of these signatures.

Single nucleotide polymorphisms at heat shock protein 90 gene and their association with thermo-tolerance potential in selected indigenous Nigerian cattle.

Heat shock protein (HSP) 90 gene provides protection and adaptation to thermal assault and certain polymorphisms have been associated to heat tolerance in humans and animals. Single nucleotide polymorphisms (SNPs) of HSP 90 gene were used to evaluate the scientific basis of heat tolerance in four zebu breeds of Nigeria. The DNA was extracted from skin tissue of 90 adult bulls representing White Fulani (WF), Sokoto Gudali (SG), Red Bororo (RB), and Ambala (AM). The SNPs were determined in DNAs using PCR, sequencing, and visualization and bio-editing by chromatogram in SeqMan Ngen tool. Subsequently, respective genotypes were constructed and genotypic and allelic frequencies were computed. Also, body parameters related to heat stress (HS) including body temperature (BT), rectal temperature (RT), and respiratory rates (RR) were taken for each animal before biological sampling and heat tolerance coefficient (HTC) was calculated. We detected four SNPs distinct/specific for each breed as follows: change from thymine (T) to guanine (G) at position 116 (T116G) in RB, G to cytosine (C) at 220 (G220C) in SG, G to adenine (A) at two positions, 346 (G346A) and 390 (G390A) in AM and WF, respectively. Heterozygous SNPs showed significantly lower values (P < 0.0001) for BT, RT, RR, and HTC than homozygous genotypes at all positions. We hypothesize that animals with heterozygous SNPs in exon 3 of HSP 90 may be tolerant to HS. These SNPs can be used as bio-markers for screening large populations of cattle for tolerance to hot tropical conditions in Nigeria and other sub-humid places.

Genomic and evolutionary aspects of chloroplast tRNA in monocot plants.

BACKGROUND: Chloroplasts are one of the most indispensable organelles that make life forms on the earth possible by their capacity to photosynthesize. These organelles possess a circular genome with a number of coding genes responsible for self-regulation. tRNAs are an important evolutionary-conserved gene family that are responsible for protein translation. However, within the chloroplast genome, tRNA machinery are poorly understood. RESULTS: In the present study, the chloroplast genome of six monocot plants, Oryza nivara (NC_005973), Oryza sativa (NC_001320), Sachharum officinarum (NC_006084), Sorghum bicolor (NC_008602), Triticum aestivum (NC_002762), and Zea mays (NC_001666) were downloaded and analyzed to identify tRNA sequences. Further analysis of the tRNA sequences in the chloroplast genomes of the monocot plants resulted in the identification of several novel features. The length of tRNAs in the chloroplast genome of the monocot plants ranged from 59 to 155 nucleotides. Pair-wise sequence alignment revealed the presence of a conserved A-C-x-U-A-x-U-A-x-U-x5-U-A-A nucleotide consensus sequence. In addition, the tRNAs in chloroplast genomes of the monocot plants also contain 21-28 anti-codons against 61 sense codons in the genome. They also contain a group I intron and a C-A-U anti-codon for tRNAIle, which is a common anti-codon of tRNAMet. Evolutionary analysis indicates that tRNAs in the chloroplast genome have evolved from multiple common ancestors, and tRNAMet appears to be the ancestral tRNA that underwent duplication and diversification to give rise to other tRNAs. CONCLUSION: The results obtained from the study of chloroplast tRNA will greatly help to increase our understanding of tRNA biology at a new level. Functional studies of the reported novel aspects of the chloroplast tRNA of the monocot plants will greatly help to decipher their roles in diverse cellular processes.

Promutagenicity of 8-Chloroguanine, A Major Inflammation-Induced Halogenated DNA Lesion.

Chronic inflammation is closely associated with cancer development. One possible mechanism for inflammation-induced carcinogenesis is DNA damage caused by reactive halogen species, such as hypochlorous acid, which is released by myeloperoxidase to kill pathogens. Hypochlorous acid can attack genomic DNA to produce 8-chloro-2'-deoxyguanosine (ClG) as a major lesion. It has been postulated that ClG promotes mutagenic replication using its syn conformer; yet, the structural basis for ClG-induced mutagenesis is unknown. We obtained crystal structures and kinetics data for nucleotide incorporation past a templating ClG using human DNA polymerase ß (polß) as a model enzyme for high-fidelity DNA polymerases. The structures showed that ClG formed base pairs with incoming dCTP and dGTP using its anti and syn conformers, respectively. Kinetic studies showed that polß incorporated dGTP only 15-fold less efficiently than dCTP, suggesting that replication across ClG is promutagenic. Two hydrogen bonds between syn-ClG and anti-dGTP and a water-mediated hydrogen bond appeared to facilitate mutagenic replication opposite the major halogenated guanine lesion. These results suggest that ClG in DNA promotes G to C transversion mutations by forming Hoogsteen base pairing between syn-ClG and anti-G during DNA synthesis.

Evidence for the Selective Basis of Transition-to-Transversion Substitution Bias in Two RNA Viruses.

The substitution rates of transitions are higher than expected by chance relative to those of transversions. Many have argued that selection disfavors transversions, as nonsynonymous transversions are less likely to conserve biochemical properties of the original amino acid. Only recently has it become feasible to directly test this selective hypothesis by comparing the fitness effects of a large number of transition and transversion mutations. For example, a recent study of six viruses and one beta-lactamase gene did not find evidence supporting the selective hypothesis. Here, we analyze the relative fitness effects of transition and transversion mutations from our recently published genome-wide study of mutational fitness effects in influenza virus. In contrast to prior work, we find that transversions are significantly more detrimental than transitions. Using what we believe to be an improved statistical framework, we also identify a similar trend in two HIV data sets. We further demonstrate a fitness difference in transition and transversion mutations using four deep mutational scanning data sets of influenza virus and HIV, which provided adequate statistical power. We find that three of the most commonly cited radical/conservative amino acid categories are predictive of fitness, supporting their utility in studies of positive selection and codon usage bias. We conclude that selection is a major contributor to the transition:transversion substitution bias in viruses and that this effect is only partially explained by the greater likelihood of transversion mutations to cause radical as opposed to conservative amino acid changes.

Mutational Biases Influence Parallel Adaptation.

While mutational biases strongly influence neutral molecular evolution, the role of mutational biases in shaping the course of adaptation is less clear. Here we consider the frequency of transitions relative to transversions among adaptive substitutions. Because mutation rates for transitions are higher than those for transversions, if mutational biases influence the dynamics of adaptation, then transitions should be overrepresented among documented adaptive substitutions. To test this hypothesis, we assembled two sets of data on putatively adaptive amino acid replacements that have occurred in parallel during evolution, either in nature or in the laboratory. We find that the frequency of transitions in these data sets is much higher than would be predicted under a null model where mutation has no effect. Our results are qualitatively similar even if we restrict ourself to changes that have occurred, not merely twice, but three or more times. These results suggest that the course of adaptation is biased by mutation.

Mutational signatures indicative of environmental stress in bacteria.

Evolutionary innovations are dependent on mutations. Mutation rates are increased by adverse conditions in the laboratory, but there is no evidence that stressful environments that do not directly impact on DNA leave a mutational imprint on extant genomes. Mutational spectra in the laboratory are normally determined with unstressed cells but are unavailable with stressed bacteria. To by-pass problems with viability, selection effects, and growth rate differences due to stressful environments, in this study we used a set of genetically engineered strains to identify the mutational spectrum associated with nutritional stress. The strain set members each had a fixed level of the master regulator protein, RpoS, which controls the general stress response of Escherichia coli. By assessing mutations in cycA gene from 485 cycloserine resistant mutants collected from as many independent cultures with three distinct perceived stress (RpoS) levels, we were able establish a dose-dependent relationship between stress and mutational spectra. The altered mutational patterns included base pair substitutions, single base pair indels, longer indels, and transpositions of different insertion sequences. The mutational spectrum of low-RpoS cells closely matches the genome-wide spectrum previously generated in laboratory environments, while the spectra of high RpoS, high perceived stress cells more closely matches spectra found in comparisons of extant genomes. Our results offer an explanation of the uneven mutational profiles such as the transition-transversion biases observed in extant genomes and provide a framework for assessing the contribution of stress-induced mutagenesis to evolutionary transitions and the mutational emergence of antibiotic resistance and disease states.

Kinetics, structure, and mechanism of 8-Oxo-7,8-dihydro-2'-deoxyguanosine bypass by human DNA polymerase η.

DNA damage incurred by a multitude of endogenous and exogenous factors constitutes an inevitable challenge for the replication machinery. Cells rely on various mechanisms to either remove lesions or bypass them in a more or less error-prone fashion. The latter pathway involves the Y-family polymerases that catalyze trans-lesion synthesis across sites of damaged DNA. 7,8-Dihydro-8-oxo-2'-deoxyguanosine (8-oxoG) is a major lesion that is a consequence of oxidative stress and is associated with cancer, aging, hepatitis, and infertility. We have used steady-state and transient-state kinetics in conjunction with mass spectrometry to analyze in vitro bypass of 8-oxoG by human DNA polymerase η (hpol η). Unlike the high fidelity polymerases that show preferential insertion of A opposite 8-oxoG, hpol η is capable of bypassing 8-oxoG in a mostly error-free fashion, thus preventing GC→AT transversion mutations. Crystal structures of ternary hpol η-DNA complexes and incoming dCTP, dATP, or dGTP opposite 8-oxoG reveal that an arginine from the finger domain assumes a key role in avoiding formation of the nascent 8-oxoG:A pair. That hpol η discriminates against dATP exclusively at the insertion stage is confirmed by structures of ternary complexes that allow visualization of the extension step. These structures with G:dCTP following either 8-oxoG:C or 8-oxoG:A pairs exhibit virtually identical active site conformations. Our combined data provide a detailed understanding of hpol η bypass of the most common oxidative DNA lesion.

Possible contribution of 8-hydroxydeoxyguanosine to gene mutations in the kidney DNA of gpt delta rats following potassium bromate treatment.

8-Hydroxydeoxyguanosine (8-OHdG) is well known not only as an effective biomarker of oxidative stress but also as a mutagenic DNA modification. Incorporation of dAMP at the opposite site of 8-OHdG induces G>T or A>C transversions. However, in vivo analyses of gene mutations caused by potassium bromate (KBrO3), which can induce 8-OHdG at carcinogenic target sites, showed that G>T was prominent in the small intestines of mice, but not in the kidneys of rats. Because KBrO3 was a much clearer carcinogen in the kidneys of rats, detailed analyses of gene mutations in the kidney DNA of rats treated with KBrO3 could improve our understanding of oxidative stress-mediated carcinogenesis. In the current study, site-specific reporter gene mutation assays were performed in the kidneys of gpt delta rats treated with KBrO3. Groups of 5 gpt delta rats were treated with KBrO3 at concentrations of 0, 125, 250, or 500 ppm in the drinking water for 9 weeks. At necropsy, the kidneys were macroscopically divided into the cortex and medulla. 8-OHdG levels in DNA extracted from the cortex were dramatically elevated at concentrations of 250 ppm and higher compared with those from the medulla. Cortex-specific increases in mutant frequencies in gpt and red/gam genes were found at 500 ppm. Mutation spectrum and sequence analyses of their mutants demonstrated significant elevations in A>T transversions in the gpt gene and single base deletions at guanine or adenine in the gpt or red/gam genes. While A>T transversions and single base deletions of adenine may result from the oxidized modification of adenine, the contribution of 8-OHdG to gene mutations was limited despite possible participation of the 8-OHdG repair process in guanine deletion.