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Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventionallacZviral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than thelacZassay. While thelacZassay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with >80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focused on 4 model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at 4 time points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS, (2) identify similarities in biological responses, (3) compare PFAS potency through benchmark concentration analysis, and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (ie, was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Transgenic rodent (TGR) models use bacterial reporter genes to quantify in vivo mutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation pattern analysis to inform mutational mechanisms. We used this approach to identify 2751 independent lacZ mutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene, N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706 lacZ mutations from eight additional environmental mutagens. We report that lacZ gene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a carcinogen present in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results suggest that the analysis of chemically induced mutations in the lacZ gene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and propose novel environmental causes of mutation signatures observed in human cancers., Marc A. Beal, Matt J. Meier et al. use a transgenic rodent model with NGS to inform mutational signature analyses. They analyze lacZ mutations from the bone marrow of MutaMouse animals exposed to chemical mutagens. They report that lacZ sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes.

Despite the widespread use of transcriptomics technologies in toxicology research, acceptance of the data by regulatory agencies to support the hazard assessment is still limited. Fundamental issues contributing to this are the lack of reproducibility in transcriptomics data analysis arising from variance in the methods used to generate data and differences in the data processing. While research applications are flexible in the way the data are generated and interpreted, this is not the case for regulatory applications where an unambiguous answer, possibly later subject to legal scrutiny, is required. A reference analysis framework would give greater credibility to the data and allow the practitioners to justify their use of an alternative bioinformatic process by referring to a standard. In this publication, we propose a method called omics data analysis framework for regulatory application (R-ODAF), which has been built as a user-friendly pipeline to analyze raw transcriptomics data from microarray and next-generation sequencing. In the R-ODAF, we also propose additional statistical steps to remove the number of false positives obtained from standard data analysis pipelines for RNA-sequencing. We illustrate the added value of R-ODAF, compared to a standard workflow, using a typical toxicogenomics dataset of hepatocytes exposed to paracetamol.

Higher-throughput, mode-of-action-based assays provide a valuable approach to expedite chemical evaluation for human health risk assessment. In this study, we combined the high-throughput alkaline DNA damage-sensing CometChip® assay with the TGx-DDI transcriptomic biomarker (DDI = DNA damage-inducing) using high-throughput TempO-Seq®, as an integrated genotoxicity testing approach. We used metabolically competent differentiated human HepaRG™ cell cultures to enable the identification of chemicals that require bioactivation to cause genotoxicity. We studied 12 chemicals (nine DDI, three non-DDI) in increasing concentrations to measure and classify chemicals based on their ability to damage DNA. The CometChip® classified 10/12 test chemicals correctly, missing a positive DDI call for aflatoxin B1 and propyl gallate. The poor detection of aflatoxin B1 adducts is consistent with the insensitivity of the standard alkaline comet assay to bulky lesions (a shortcoming that can be overcome by trapping repair intermediates). The TGx-DDI biomarker accurately classified 10/12 agents. TGx-DDI correctly identified aflatoxin B1 as DDI, demonstrating efficacy for combined used of these complementary methodologies. Zidovudine, a known DDI chemical, was misclassified as it inhibits transcription, which prevents measurable changes in gene expression. Eugenol, a non-DDI chemical known to render misleading positive results at high concentrations, was classified as DDI at the highest concentration tested. When combined, the CometChip® assay and the TGx-DDI biomarker were 100% accurate in identifying chemicals that induce DNA damage. Quantitative benchmark concentration (BMC) modeling was applied to evaluate chemical potencies for both assays. The BMCs for the CometChip® assay and the TGx-DDI biomarker were highly concordant (within 4-fold) and resulted in identical potency rankings. These results demonstrate that these two assays can be integrated for efficient identification and potency ranking of DNA damaging agents in HepaRG™ cell cultures.

The MutaMouse transgenic rodent model is widely used for assessing in vivo mutagenicity. Here, we report the characterization of MutaMouse’s whole genome sequence and its genetic variants compared to the C57BL/6 reference genome. High coverage (>50X) next-generation sequencing (NGS) of whole genomes from multiple MutaMouse animals from the Health Canada (HC) colony showed ~5 million SNVs per genome, ~20% of which are putatively novel. Sequencing of two animals from a geographically separated colony at Covance indicated that, over the course of 23 years, each colony accumulated 47,847 (HC) and 17,677 (Covance) non-parental homozygous single nucleotide variants. We found no novel nonsense or missense mutations that impair the MutaMouse response to genotoxic agents. Pairing sequencing data with array comparative genomic hybridization (aCGH) improved the accuracy and resolution of copy number variants (CNVs) calls and identified 300 genomic regions with CNVs. We also used long-read sequence technology (PacBio) to show that the transgene integration site involved a large deletion event with multiple inversions and rearrangements near a retrotransposon. The MutaMouse genome gives important genetic context to studies using this model, offers insight on the mechanisms of structural variant formation, and contributes a framework to analyze aCGH results alongside NGS data.

Polycyclic aromatic hydrocarbons like benzo[a]pyrene (BaP) are ubiquitous environmental contaminants formed during incomplete combustion of organic materials. Our prior work showed that transplacental exposure to BaP depletes ovarian follicles and increases prevalence of epithelial ovarian tumors later in life. We used the MutaMouse transgenic rodent model to address the hypothesis that ovarian mutations play a role in tumorigenesis caused by prenatal exposure to BaP. Pregnant MutaMouse females were treated with 0, 10, 20, or 40 mg/(kg day) BaP orally on gestational days 7-16, covering critical windows of ovarian development. Female offspring were euthanized at 10 weeks of age; some ovaries with oviducts were processed for follicle counting; other ovaries/oviducts and bone marrow were processed for determination of lacZ mutant frequency (MF). Mutant plaques were pooled within dose groups and sequenced to determine the mutation spectrum. BaP exposure caused highly significant dose-related decreases in ovarian follicles and increases in ovarian/oviductal and bone marrow mutant frequencies at all doses. Absence of follicles, cell packets, and epithelial tubular structures were observed with 20 and 40 mg/(kg day) BaP. Depletion of ovarian germ cells was inversely associated with ovarian MF. BaP induced primarily G > T and G > C transversions and deletions in ovaries/oviducts and bone marrow cells and produced a mutation signature highly consistent with that of tobacco smoking in human cancers. Overall, our results show that prenatal BaP exposure significantly depletes ovarian germ cells, causes histopathological abnormalities, and increases the burden of ovarian/oviductal mutations, which may be involved in pathogenesis of epithelial ovarian tumors. Environ. Mol. Mutagen. 60:410-420, 2019. © 2018 Her Majesty the Queen in Right of Canada.

Changes in the mammalian gut microbiome are linked to the impairment of immunological function and numerous other pathologies. Antimicrobial silver nanoparticles (AgNPs) are incorporated into numerous consumer products (e.g., clothing, cosmetics, food packaging), which may directly impact the gut microbiome through ingestion. The human health impact of chronic AgNP ingestion is still uncertain, but evidence from exposure to other antimicrobials provides a strong rationale to assess AgNP effects on organ function, immunity, metabolism, and gut-associated microbiota. To investigate this, mice were gavaged daily for 5 weeks with saline, AgNPs, antibiotics (ciprofloxacin and metronidazole), or AgNPs combined with antibiotics. Animals were weighed daily, assessed for glucose tolerance, organ function, tissue and blood cytokine and leukocyte levels. At the end of the study, we used 16S rDNA amplicon and whole-metagenome shotgun sequencing to assess changes in the gut microbiome. In mice exposed to both AgNPs and antibiotics, silver was found in the stomach, and small and large intestines, but negligible amounts were present in other organs examined. Mice exposed to AgNPs alone showed minimal tissue silver levels. Antibiotics, but not AgNPs, altered glucose metabolism. Mice given AgNPs and antibiotics together demonstrated slower weight gain, reduced peripheral lymphocytes, and elevated splenic, but not circulatory markers of inflammation. 16S rDNA profiling of cecum and feces and metagenomic sequencing of fecal DNA demonstrated that combined AgNP-antibiotic treatment also significantly altered the structure and function of the gut microbiota, including depletion of the indicator species Akkermansia muciniphila. This study provides evidence for possible biological effects from repeated ingestion of AgNP-containing consumer products when antibiotics are also being used and raises concern that an impaired gut microbiome (e.g., through antibiotic use) can potentiate the harm from chemical exposures such as AgNPs.

Per- and poly-fluoroalkyl substances (PFAS) are widely found in the environment because of their extensive use and persistence. Although several PFAS are well studied, most lack toxicity data to inform human health hazard and risk assessment. This study focussed on four model PFAS: perfluorooctanoic acid (PFOA; 8 carbon), perfluorobutane sulfonate (PFBS; 4 carbon), perfluorooctane sulfonate (PFOS; 8 carbon), and perfluorodecane sulfonate (PFDS; 10 carbon). Human primary liver cell spheroids (pooled from 10 donors) were exposed to 10 concentrations of each PFAS and analyzed at four time-points. The approach aimed to: (1) identify gene expression changes mediated by the PFAS; (2) identify similarities in biological responses; (3) compare PFAS potency through benchmark concentration analysis; and (4) derive bioactivity exposure ratios (ratio of the concentration at which biological responses occur, relative to daily human exposure). All PFAS induced transcriptional changes in cholesterol biosynthesis and lipid metabolism pathways, and predicted PPARα activation. PFOS exhibited the most transcriptional activity and had a highly similar gene expression profile to PFDS. PFBS induced the least transcriptional changes and the highest benchmark concentration (i.e., was the least potent). The data indicate that these PFAS may have common molecular targets and toxicities, but that PFOS and PFDS are the most similar. The transcriptomic bioactivity exposure ratios derived here for PFOA and PFOS were comparable to those derived using rodent apical endpoints in risk assessments. These data provide a baseline level of toxicity for comparison with other known PFAS using this testing strategy.

Transformative advances in metagenomics are providing an unprecedented ability to characterize the enormous diversity of microorganisms and invertebrates sustaining soil health and water quality. These advances are enabling a better recognition of the ecological linkages between soil and water, and the biodiversity exchanges between these two reservoirs. They are also providing new perspectives for understanding microorganisms and invertebrates as part of interacting communities (i.e. microbiomes and zoobiomes), and considering plants, animals, and humans as holobionts comprised of their own cells as well as diverse microorganisms and invertebrates often acquired from soil and water. The Government of Canada's Genomics Research and Development Initiative (GRDI) launched the Ecobiomics Project to coordinate metagenomics capacity building across federal departments, and to apply metagenomics to better characterize microbial and invertebrate biodiversity for advancing environmental assessment, monitoring, and remediation activities. The Project has adopted standard methods for soil, water, and invertebrate sampling, collection and provenance of metadata, and nucleic acid extraction. High-throughput sequencing is located at a centralized sequencing facility. A centralized Bioinformatics Platform was established to enable a novel government-wide approach to harmonize metagenomics data collection, storage and bioinformatics analyses. Sixteen research projects were initiated under Soil Microbiome, Aquatic Microbiome, and Invertebrate Zoobiome Themes. Genomic observatories were established at long-term environmental monitoring sites for providing more comprehensive biodiversity reference points to assess environmental change.

Background: Mosaicism, the presence of genetically distinct cell populations within an organism, has emerged as an important contributor to disease. Mutational events occurring during embryonic development can cause mosaicism in any tissue, but the influence of environmental factors on levels of mosaicism is unclear. Objectives: We investigated whether in utero exposure to the widespread environmental mutagen benzo[a]pyrene (BaP) has an impact on the burden and distribution of mutations in adult mice. Methods: We used the Muta™Mouse transgenic rodent model to quantify and characterize mutations in the offspring of pregnant mice exposed to BaP during postconception days 7 through 16, covering the major period of organogenesis in mice. Next-generation DNA sequencing was then used to determine the spectrum of mutations induced in adult mice that were exposed to BaP during fetal development. Results: Mutation frequency was significantly increased in the bone marrow, liver, brain, and sperm of first filial generation (F1) males. Developing embryos accumulated more mutations and exhibited higher proportions of mosaicism than exposed adults, particularly in the brain. Decreased sperm count and motility revealed additional negative impacts on the reproductive function of F1 males. Conclusion: In utero exposure to environmental mutagens contributes to somatic and germline mosaicism, permanently affecting both the genetic health of the F1 and the population gene pool. Citation: Meier MJ, O’Brien JM, Beal MA, Allan B, Yauk CL, Marchetti F. 2017. In utero exposure to benzo[a]pyrene increases mutation burden in the soma and sperm of adult mice. Environ Health Perspect 125:82–88; http://dx.doi.org/10.1289/EHP211

Transgenic rodent (TGR) models use bacterial reporter genes to quantifyin vivomutagenesis. Pairing TGR assays with next-generation sequencing (NGS) enables comprehensive mutation spectrum analysis to inform mutational mechanisms. We used this approach to identify 2,751 independentlacZmutations in the bone marrow of MutaMouse animals exposed to four chemical mutagens: benzo[a]pyrene,N-ethyl-N-nitrosourea, procarbazine, and triethylenemelamine. We also collected published data for 706lacZmutations from eight additional environmental mutagens. We demonstrate thatlacZgene sequencing generates chemical-specific mutation signatures observed in human cancers with established environmental causes. For example, the mutation signature of benzo[a]pyrene, a potent carcinogen in tobacco smoke, matched the signature associated with tobacco-induced lung cancers. Our results show that the analysis of chemically induced mutations in thelacZgene shortly after exposure provides an effective approach to characterize human-relevant mechanisms of carcinogenesis and identify novel environmental causes of mutation signatures observed in human cancers.

Understanding the effects of environmental exposures on germline mutation rates has been a decades-long pursuit in genetics. We used next-generation sequencing and comparative genomic hybridization arrays to investigate genome-wide mutations in the offspring of male mice exposed to benzo(a)pyrene (BaP), a common environmental pollutant. We demonstrate that offspring developing from sperm exposed during the mitotic or post-mitotic phases of spermatogenesis have significantly more de novo single nucleotide variants (1.8-fold; P, Marc Beal et al. investigate the genomic consequences of paternal exposure to benzo(a)pyrene (BaP), a common carcinogenic pollutant found in car exhaust fumes, tobacco smoke and grilled meats. They find that mouse offspring from males exposed to BaP have more de novo mutations, suggesting BaP exposure in males is associated with adverse health outcomes in offspring.

Metagenomics allows the study of genes related to xenobiotic degradation in a culture-independent manner, but many of these studies are limited by the lack of genomic context for metagenomic sequences. This study combined a phenotypic screen known as substrate-induced gene expression (SIGEX) with whole-metagenome shotgun sequencing. SIGEX is a high-throughput promoter-trap method that relies on transcriptional activation of a green fluorescent protein (GFP) reporter gene in response to an inducing compound and subsequent fluorescence-activated cell sorting to isolate individual inducible clones from a metagenomic DNA library. We describe a SIGEX procedure with improved library construction from fragmented metagenomic DNA and improved flow cytometry sorting procedures. We used SIGEX to interrogate an aromatic hydrocarbon (AH)-contaminated soil metagenome. The recovered clones contained sequences with various degrees of similarity to genes (or partial genes) involved in aromatic metabolism, for example, nahG (salicylate oxygenase) family genes and their respective upstream nahR regulators. To obtain a broader context for the recovered fragments, clones were mapped to contigs derived from de novo assembly of shotgun-sequenced metagenomic DNA which, in most cases, contained complete operons involved in aromatic metabolism, providing greater insight into the origin of the metagenomic fragments. A comparable set of contigs was generated using a significantly less computationally intensive procedure in which assembly of shotgun-sequenced metagenomic DNA was directed by the SIGEX-recovered sequences. This methodology may have broad applicability in identifying biologically relevant subsets of metagenomes (including both novel and known sequences) that can be targeted computationally by in silico assembly and prediction tools.

Anthropogenic activities can disrupt soil ecosystems, normally resulting in reduced soil microbial health. Regulatory agencies need to determine the effects of uncharacterized substances on soil microbial health to establish the safety of these chemicals if they end up in the environment. Previous work has focused on measuring traditional ecotoxicologial endpoints within the categories of microbial biomass, activity, and community structure/diversity. Because these tests can be labor intensive, lengthy to conduct, and cannot measure changes in individual gene functions, we wanted to establish whether metatranscriptomics could be used as a more sensitive endpoint and provide a perspective on community function that is more informative than taxonomic identification of microbes alone. We spiked a freshly collected sandy loam soil (Vulcan, Alberta, Canada) with 0, 60, 145, 347, 833, and 2000 mg kg−1 of silver nanoparticles (AgNPs), a known antagonist of microorganisms due to its propensity for dissolution of toxic silver ions. Assessments performed in our previous work using traditional tests demonstrated the toxicity of AgNPs on soil microbial processes. We expanded this analysis with genomics-based tests by measuring changes in community taxonomic structure and function using 16S rDNA profiling and metatranscriptomics. In addition to identifying bacterial taxa affected by AgNPs, we found that genes involved in heavy metal resistance (e.g., the CzcA efflux pump) and other toxicity response pathways were highly upregulated in the presence of silver. Dose-response analysis using BMDExpress2 software successfully modeled many physiologically relevant genes responding to low concentrations of AgNPs. We found that the transcriptomic point of departure (BMD50) was lower than the IC50s calculated using the traditional tests in our previous work. These results suggest that dose-response modeling of metatranscriptomic gene expression is a useful tool in soil microbial health assessment. Summary Genomics-based endpoints for the assessment of soil microbial health can be used to perform quantitative dose-response modeling, and soil-based RNAseq adds functional insights.

Bacillus amyloliquefaciens NRRL 942 is a Gram-positive bacterium with several potential industrial uses. We have sequenced the whole genome of this organism to assist in understanding the biological mechanisms that might modulate human health or environmental risk in the event of its release into the environment.

Pseudomonas fluorescens is a Gram-negative bacterium with versatile metabolic functions and potential industrial uses. We sequenced P. fluorescens strain ATCC 13525 with the goal of determining virulence factors and antibiotic resistance genes to predict the potential impacts on human and environmental health in the event of exposure.

No study has evaluated the mutagenicity of atmospheres with a calculated air quality health index (AQHI). Thus, we generated in a UV-light-containing reaction chamber two simulated atmospheres (SAs) with similar AQHIs but different proportions of criteria pollutants and evaluated them for mutagenicity in three Salmonella strains at the air-agar interface. We continuously injected into the chamber gasoline, nitric oxide, and ammonium sulfate, as well as either α-pinene to produce SA-PM, which had a high concentration of particulate matter (PM): 119 ppb ozone (O(3)), 321 ppb NO(2), and 1007 μg/m(3) PM(2.5); or isoprene to produce SA-O(3), which had a high ozone (O(3)) concentration: 415 ppb O(3), 633 ppb NO(2), and 55 μg/m(3) PM(2.5). Neither PM(2.5) extracts, NO(2), or O(3) alone, nor non-photo-oxidized mixtures were mutagenic or cytotoxic. Both photo-oxidized atmospheres were largely direct-acting base-substitution mutagens with similar mutagenic potencies in TA100 and TA104. The mutagenic potencies [(revertants/h)/(mgC/m(3))] of SA-PM (4.3 ± 0.4) and SA-O(3) (9.5 ± 1.3) in TA100 were significantly different (P < 0.0001), but the mutation spectra were not (P = 0.16), being ~54% C → T and ~46% C → A. Thus, the AQHI may have some predictive value for the mutagenicity of the gas phase of air.

Microbial based cleaning products (MBCPs) are a new generation of cleaning products that are gaining greater use in household, institutional, and industrial settings. Little is known about the exact microbial composition of these products because they are not identified in detail on product labels and formulations are often proprietary. To gain a better understanding of their microbial and fungal composition towards risk assessment, the cultivable microorganisms and rDNA was surveyed for microbial content in five different MBCPs manufactured and sold in North America. Individual bacterial and fungal colonies were identified by ribosequencing and fatty acid methyl ester (FAME) gas chromatography. Metagenomic DNA (mDNA) corresponding to each of the products was subjected to amplification and short read sequencing of seven of the variable regions of the bacterial 16S ribosomal DNA. Taken together, the cultivable microorganism and rDNA survey analyses showed that three of the products were simple mixtures of Bacillus species. The two other products featured a mixture of cultivable fungi with Bacilli, and by rDNA survey analysis, they featured greater microbial complexity. This study improves our understanding of the microbial composition of several MBCPs towards a more comprehensive risk assessment.

During bacterial replication, DNA polymerases may encounter DNA lesions that block processive DNA synthesis. Uncoupling the replicative helicase from the stalled DNA polymerase results in the formation of single-stranded DNA (ssDNA) gaps, which are repaired by postreplication repair (PRR), a process that involves at least three mechanisms that collectively remove, circumvent or bypass lesions. RecA mediated excision repair (RAMER) and homologous recombination (HR) are strand-exchange mechanisms that appear to be the predominant strategies for gap repair in the absence of prolonged SOS induction. During RAMER, RecA mediates pairing of damaged ssDNA with an undamaged homologous duplex and subsequent exchange of strands between the damaged and undamaged DNA. Repair of the lesion occurs in the context of the strand-exchange product and is initiated by UvrABC excinuclease; the resulting patch is filled by DNA synthesis using the complementary strand of the homologous duplex as a template. HR uses a complementary strand of an undamaged homologous duplex as a transient template for DNA synthesis. HR requires the formation and resolution of Holliday junctions, and is a mechanism to circumvent the lesion; lesions persisting in one of the daughter DNA duplexes will normally be repaired prior to subsequent rounds of replication/cell division. Translesion DNA Synthesis (TLS) does not involve strand-exchange mechanisms; it is carried out by specialized DNA polymerases that are able to catalyze nucleotide incorporation opposite lesions that cannot be bypassed by high-fidelity replicative polymerases. Maximum levels of TLS occur during prolonged SOS induction generally associated with increased mutagenesis. RAMER, HR and TLS are alternative mechanisms for processing a common intermediate-the ssDNA gap containing a RecA nucleofilament. The actual pathway that is utilized will be strongly influenced by multiple factors, including the blocking/coding capacity of the lesion, the nature of the gene products that can be assembled at the ssDNA gap, the availability of a homologous partner for RAMER and HR, and protein:protein interactions and post-translational modifications that modulate the mutagenic activity of Pol-IV and Pol-V.

From C Fund Conf no Corre D C J Am 0190 a 20 doi:1 A 58-year-old white man presented with a 10-year history of a progressive eruption that began on his scalp, resulting in widespread alopecic patches, and gradually spread to involve his trunk and extremities. He was otherwise healthy. Fig 1 shows representative lesions on the scalp and flank. Histologic findings from biopsies obtained from lesions on the trunk are shown in Fig 2.