In Vitro Gamma Mutagenesis Techniques in Rice (Oryza sativa L. var. Lazarroz FL).

Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.

The cold - resistance mechanism of a mutagenic Volvariella volvacea strain VH3 with outstanding traits revealed by transcriptome profiling.

BACKGROUND: The straw mushroom (Volvariella volvacea) is one of the important vegetables that is popular for its delicious taste. However, the straw mushroom is sensitive to low temperature, resulting in economic loss during transportation and storage. We obtained a novel straw mushroom strain, named VH3, via ultraviolet mutagenesis. RESULTS: Our study revealed that VH3 exhibited high cold resistance compared to an ordinary straw mushroom cultivar, V23. We found that the electrolyte leakages of VH3 were always significantly lower than that of V23 treated with 4 °C for 0 h, 2 h,4 h, 8 h, 16 h, and 24 h. Before cold treatment (0 h), there were no difference of MDA contents, SOD activities, and CAT activities between VH3 and V23. At the late stage (8 h, 26 h, and 24 h) of cold treatment, the MDA contents of VH3 were lower while both the SOD and CAT activities were higher than those of V23. To investigate the potential mechanisms of VH3 cold resistance, we performed transcriptome sequencing to detect the transcriptome profiling of VH3 and V23 after 0 h and 4 h cold treatment. Transcriptome sequencing revealed that 111 differentially expressed genes (DEG) between V23 (0 h) and VH3 (0 h) (V23-0_vs_VH3-0), consisting 50 up-regulated and 61 down-regulated DEGs. A total of 117 DEGs were obtained between V23 (4 h) and VH3(4 h) (V23-4_vs_VH3-4), containing 94 up-regulated and 23 down-regulated DEGs. Among these DEGs, VVO_00021 and VVO_00017 were up-regulated while VVO_00003, VVO_00004, VVO_00010, and VVO_00030 were down-regulated in V23-0_vs_VH3-0 and VH3-4_vs_V23-4. KEGG and GO analysis revealed that the 6 DEGs were annotated to pathways related to cold stress. Besides, the GA3 content was also decreased in VH3. CONCLUSIONS: Collectively, our study first revealed that the increased cold resistance of VH3 might be caused by the expression change of VVO_00003, VVO_00004, VVO_00017, VVO_00021, and VVO_00030, and decreased GA3.

UVB mutagenesis differs in Nras- and Braf-mutant mouse models of melanoma.

BRAF-mutant melanomas are more likely than NRAS-mutant melanomas to arise in anatomical locations protected from chronic sun damage. We hypothesized that this discrepancy in tumor location is a consequence of the differential sensitivity of BRAF and NRAS-mutant melanocytes to ultraviolet light (UV)-mediated carcinogenesis. We tested this hypothesis by comparing the mutagenic consequences of a single neonatal, ultraviolet-AI (UVA; 340-400 nm) or ultraviolet-B (UVB; 280-390 nm) exposure in mouse models heterozygous for mutant Braf or homozygous for mutant Nras Tumor onset was accelerated by UVB, but not UVA, and the resulting melanomas contained recurrent mutations affecting the RING domain of MAP3K1 and Actin-binding domain of Filamin A. Melanomas from UVB-irradiated, Braf-mutant mice averaged twice as many single-nucleotide variants and five times as many dipyrimidine variants than tumors from similarly irradiated Nras-mutant mice. A mutational signature discovered in UVB-accelerated tumors mirrored COSMIC signatures associated with human skin cancer and was more prominent in Braf- than Nras-mutant murine melanomas. These data show that a single UVB exposure yields a greater burden of mutations in murine tumors driven by oncogenic Braf.

Applied mutagenesis could improve economically important traits in bitter gourd (Momordica charantia L.).

Mutants with unique characters have played a key role in discovery of gene, mapping, functional genomics and breeding in many vegetable crops, but information on bitter gourd is lacking. Induction of mutation by gamma rays (Co60 source) at five different doses (50 Gy, 100 Gy, 150 Gy, 200 Gy and 250 Gy) was studied in four widely divergent bitter gourd genotypes BG-1346501, Meghna-2, Special Boulder and Selection-1 in M1 generation. Reduction in seed germination percentage, vine length and pollen fertility occurred in M1 generation with the increasing doses of mutagens. LD50 dose for BG-1346501, Meghna-2, Special Boulder and Selection-1 corresponded to 290.76 Gy, 206.12 Gy, 212.81 Gy and 213.49 Gy ᵞ radiation, respectively suggested low to medium doses (200-250 Gy) of gamma rays would be helpful in producing useful and exploitable mutants for further breeding. No remarkable effect of ᵞ radiation on fruit physicochemical characters in M1 generation were observed. M2 generation, raised from two widely divergent genotypes, BG-1346501 and Meghna-2, were screened critically and observed no significant reduction in seed germination and pollen viability, however little damage occurred particularly in vine length. There is possibility of isolating segregates in M2 generation with enhanced nutrient contents at low radiation dose. Highest mutation frequency resulted by treating Meghna-2 at 200 Gy and BG-1346501 at 100 Gy. Both genotype and mutagenic doses influenced mutagenic effectiveness. Spectrum of mutation was very low; number of putative mutants isolated from M2 generation was five in Meghna-2 and three in BG-1346501. Among six putative macro mutants isolated from M3 generation, we could identify two putative mutants, namely Meghna-2 with gynoecious sex form and BG-1346501 with high charantin, appreciable ß-carotene and high ascorbic acid contents having ample promise for further utilization in bitter gourd breeding after critical testing in subsequent generations for estimation of genetic gain and trait heritability to confirm the mutant stability.

Radiotherapy is associated with a deletion signature that contributes to poor outcomes in patients with cancer.

Ionizing radiation causes DNA damage and is a mainstay for cancer treatment, but understanding of its genomic impact is limited. We analyzed mutational spectra following radiotherapy in 190 paired primary and recurrent gliomas from the Glioma Longitudinal Analysis Consortium and 3,693 post-treatment metastatic tumors from the Hartwig Medical Foundation. We identified radiotherapy-associated significant increases in the burden of small deletions (5-15 bp) and large deletions (20+ bp to chromosome-arm length). Small deletions were characterized by a larger span size, lacking breakpoint microhomology and were genomically more dispersed when compared to pre-existing deletions and deletions in non-irradiated tumors. Mutational signature analysis implicated classical non-homologous end-joining-mediated DNA damage repair and APOBEC mutagenesis following radiotherapy. A high radiation-associated deletion burden was associated with worse clinical outcomes, suggesting that effective repair of radiation-induced DNA damage is detrimental to patient survival. These results may be leveraged to predict sensitivity to radiation therapy in recurrent cancer.

Proof of the triple prerequisite conditions which are essential for carbapenem resistance development in Klebsiella pneumoniae by using radiation-mediated mutagenesis.

Evolution of multi-drug resistant bacteria has led to worldwide research to better understand the various resistance mechanisms in these strains. Every year, novel information on carbapenem resistance and its mechanisms is being discovered. In this study, radiation-mediated mutagenesis was used to transform a carbapenem-resistant Klebsiella pneumoniae strain to a carbapenem-susceptible bacterium. Through this process, we proved three conditions of loss of the OmpK35 and the OmpK36 genes and acquisition of blaCMY-10 worked together to produce carbapenem resistance in K. pneumoniae. Loss of only one of the porins did not evoke carbapenem resistance. This is the first report on the essential contribution of these three components of carbapenem resistance in K. pneumoniae.

Developmental delay with hypotrophy associated with homozygous functionally relevant REV3L variant.

REV3L encodes a catalytic subunit of DNA polymerase zeta (Pol zeta) which is essential for the tolerance of DNA damage by inducing translesion synthesis (TLS). So far, the only Mendelian disease associated with REV3L was Moebius syndrome (3 patients with dominant REV3L mutations causing monoallelic loss-of-function were reported). We describe a homozygous ultra-rare REV3L variant (T2753R) identified with whole exome sequencing in a child without Moebius syndrome but with developmental delay, hypotrophy, and dysmorphic features who was born to healthy parents (heterozygous carriers of the variant). The variant affects the amino acid adjacent to functionally important KKRY motif. By introducing an equivalent mutation (S1192R) into the REV3 gene in yeasts, we showed that, whereas it retained residual function, it caused clear dysfunction of TLS in the nucleus and instability of mitochondrial genetic information. In particular, the mutation increased UV sensitivity measured by cell survival, decreased both the spontaneous (P < 0.005) and UV-induced (P < 0.0001) mutagenesis rates of nuclear DNA and increased the UV-induced mutagenesis rates of mitochondrial DNA (P < 0.0005). We propose that our proband is the first reported case of a REV3L associated disease different from Moebius syndrome both in terms of clinical manifestations and inheritance (autosomal recessive rather than dominant). KEY MESSAGES: First description of a human recessive disorder associated with a REV3L variant. A study in yeast showed that the variant affected the enzymatic function of the protein. In particular, it caused increased UV sensitivity and abnormal mutagenesis rates.

UV-exposure, endogenous DNA damage, and DNA replication errors shape the spectra of genome changes in human skin.

Human skin is continuously exposed to environmental DNA damage leading to the accumulation of somatic mutations over the lifetime of an individual. Mutagenesis in human skin cells can be also caused by endogenous DNA damage and by DNA replication errors. The contributions of these processes to the somatic mutation load in the skin of healthy humans has so far not been accurately assessed because the low numbers of mutations from current sequencing methodologies preclude the distinction between sequencing errors and true somatic genome changes. In this work, we sequenced genomes of single cell-derived clonal lineages obtained from primary skin cells of a large cohort of healthy individuals across a wide range of ages. We report here the range of mutation load and a comprehensive view of the various somatic genome changes that accumulate in skin cells. We demonstrate that UV-induced base substitutions, insertions and deletions are prominent even in sun-shielded skin. In addition, we detect accumulation of mutations due to spontaneous deamination of methylated cytosines as well as insertions and deletions characteristic of DNA replication errors in these cells. The endogenously induced somatic mutations and indels also demonstrate a linear increase with age, while UV-induced mutation load is age-independent. Finally, we show that DNA replication stalling at common fragile sites are potent sources of gross chromosomal rearrangements in human cells. Thus, somatic mutations in skin of healthy individuals reflect the interplay of environmental and endogenous factors in facilitating genome instability and carcinogenesis.

Meiotic susceptibility for induction of sperm with chromosomal aberrations in patients receiving combination chemotherapy for Hodgkin lymphoma.

Improvements in survival rates with gonad-sparing protocols for childhood and adolescence cancer have increased the optimism of survivors to become parents after treatment. Findings in rodents indicate that chromosomal aberrations can be induced in male germ cells by genotoxic exposures and transmitted to offspring and future generations with effects on development, fertility and health. Thus, there is a need for effective technologies to identify human sperm carrying chromosomal aberrations to assess the germ-line risks, especially for cancer survivors who have received genotoxic therapies. The time-dependent changes in the burden of sperm carrying structural chromosomal aberrations were assessed for the first time in a cancer setting, using the AM8 sperm FISH protocol which simultaneously detects abnormalities in chromosomal structure and number in sperm. Nine Hodgkin lymphoma (HL) patients provided 20 semen samples before, during, and after NOVP therapy (Novantrone, Oncovin, Velban and Prednisone) and radiation therapy that produced scattered gonadal doses from <0.05 to 0.6 Gy. Late meiosis was found to be the most sensitive to NOVP treatment for the production of sperm with chromosomal abnormalities, both in structure and number. Earlier stages of spermatogenesis were less sensitive and there was no evidence that therapy-exposed stem cells resulted in increased frequencies of sperm with abnormalities in chromosomal structure or number. This indicates that NOVP therapy may increase the risks for paternal transmission of chromosomal structural aberrations for sperm produced 32 to 45 days after a treatment with these drugs and implies that there are no excess risks for pregnancies conceived more than 6 months after this therapy. This clinical evaluation of the AM8 sperm FISH protocol indicates that it is a promising tool for assessing an individual's burden of sperm carrying chromosomal structural aberrations as well as aneuploidies after cancer therapy, with broad applications in other clinical and environmental situations that may pose aneugenic or clastogenic risks to human spermatogenesis.

Histone H4 LRS mutations can attenuate UV mutagenesis without affecting PCNA ubiquitination or sumoylation.

UV is a significant environmental agent that damages DNA. Translesion synthesis (TLS) is a DNA damage tolerance pathway that utilizes specialized DNA polymerases to replicate through the damaged DNA, often leading to mutagenesis. In eukaryotic cells, genomic DNA is organized into chromatin that is composed of nucleosomes. To date, if and/or how TLS is regulated by a specific nucleosome feature has been undocumented. We found that mutations of multiple histone H4 residues mostly or entirely embedded in the nucleosomal LRS (loss of ribosomal DNA-silencing) domain attenuate UV mutagenesis in Saccharomyces cerevisiae. The attenuation is not caused by an alteration of ubiquitination or sumoylation of PCNA (proliferating cell nuclear antigen), the modifications well-known to regulate TLS. Also, the attenuation is not caused by decreased chromatin accessibility, or by alterations of methylation of histone H3 K79, which is at the center of the LRS surface. The attenuation may result from compromised TLS by both DNA polymerases ζ and η, in which Rad6 and Rad5 are but Rad18 is not implicated. We propose that a feature of the LRS is recognized or accessed by the TLS machineries either during/after a nucleosome is disassembled in front of a lesion-stalled replication fork, or during/before a nucleosome is reassembled behind a lesion-stalled replication fork.

Using Relaxation Time to characterize biological effects of different mutagens.

All kinds of mutagenic factors may cause physiological, biochemical and genetic changes of all organisms. To characterize their characteristic biology effects, the concept of Relaxation Time (RT) was introduced for the first time, and the specific process was as follows. After mutation of organisms, the offsprings will be continuingly cultured (or cultivated) to the next generation (Rx). Once a biological effect began to show no significant difference compared to the untreated controls, the Rx was defined as the RT of the effect. In this paper, three kinds of mutagenic factors were selected to treat the seeds or seedlings of Astragalus sinicus L., subsequently, the corresponding RT was calibrated. The results showed that the RT was diverse not only among different biological effects but also among different mutagenic factors. For the RT of chemical mutagens and gamma rays, most of which are concentrated on R1, whereas the heavy ion beams have significant differences among different tracks. Among biological effects, the SOD activity and superoxide anion free radical content in the Peak region are more prominent, and their RT reaches R3 and R4, respectively. Thus, the RT may characterize the characteristic biological effects from differently mutagenic factors.

An antiphage Escherichia coli mutant for higher production of L-threonine obtained by atmospheric and room temperature plasma mutagenesis.

Phage infection is common during the production of L-threonine by E. coli, and low L-threonine production and glucose conversion percentage are bottlenecks for the efficient commercial production of L-threonine. In this study, 20 antiphage mutants producing high concentration of L-threonine were obtained by atmospheric and room temperature plasma (ARTP) mutagenesis, and an antiphage E. coli variant was characterized that exhibited the highest production of L-threonine Escherichia coli ([E. coli] TRFC-AP). The elimination of fhuA expression in E. coli TRFC-AP was responsible for phage resistance. The biomass and cell growth of E. coli TRFC-AP showed no significant differences from those of the parent strain (E. coli TRFC), and the production of L-threonine (159.3 g L-1 ) and glucose conversion percentage (51.4%) were increased by 10.9% and 9.1%, respectively, compared with those of E. coli TRFC. During threonine production (culture time of 20 h), E. coli TRFC-AP exhibited higher activities of key enzymes for glucose utilization (hexokinase, glucose phosphate dehydrogenase, phosphofructokinase, phosphoenolpyruvate carboxylase, and PYK) and threonine synthesis (glutamate synthase, aspartokinase, homoserine dehydrogenase, homoserine kinase and threonine synthase) compared to those of E. coli TRFC. The analysis of metabolic flux distribution indicated that the flux of threonine with E. coli TRFC-AP reached 69.8%, an increase of 16.0% compared with that of E. coli TRFC. Overall, higher L-threonine production and glucose conversion percentage were obtained with E. coli TRFC-AP due to increased activities of key enzymes and improved carbon flux for threonine synthesis.

In vivo functional screening for systems-level integrative cancer genomics.

With the genetic portraits of all major human malignancies now available, we next face the challenge of characterizing the function of mutated genes, their downstream targets, interactions and molecular networks. Moreover, poorly understood at the functional level are also non-mutated but dysregulated genomes, epigenomes or transcriptomes. Breakthroughs in manipulative mouse genetics offer new opportunities to probe the interplay of molecules, cells and systemic signals underlying disease pathogenesis in higher organisms. Herein, we review functional screening strategies in mice using genetic perturbation and chemical mutagenesis. We outline the spectrum of genetic tools that exist, such as transposons, CRISPR and RNAi and describe discoveries emerging from their use. Genome-wide or targeted screens are being used to uncover genomic and regulatory landscapes in oncogenesis, metastasis or drug resistance. Versatile screening systems support experimentation in diverse genetic and spatio-temporal settings to integrate molecular, cellular or environmental context-dependencies. We also review the combination of in vivo screening and barcoding strategies to study genetic interactions and quantitative cancer dynamics during tumour evolution. These scalable functional genomics approaches are transforming our ability to interrogate complex biological systems.

Cytotoxicity and Mutagenicity of Narrowband UVB to Mammalian Cells.

Phototherapy using narrowband ultraviolet-B (NB-UVB) has been shown to be more effective than conventional broadband UVB (BB-UVB) in treating a variety of skin diseases. To assess the difference in carcinogenic potential between NB-UVB and BB-UVB, we investigated the cytotoxicity via colony formation assay, genotoxicity via sister chromatid exchange (SCE) assay, mutagenicity via hypoxanthine phosphoribosyltransferase (HPRT) mutation assay, as well as cyclobutane pyrimidine dimer (CPD) formation and reactive oxygen species (ROS) generation in Chinese hamster ovary (CHO) and their NER mutant cells. The radiation dose required to reduce survival to 10% (D10 value) demonstrated BB-UVB was 10 times more cytotoxic than NB-UVB, and revealed that NB-UVB also induces DNA damage repaired by nucleotide excision repair. We also found that BB-UVB more efficiently induced SCEs and HPRT mutations per absorbed energy dosage (J/m2) than NB-UVB. However, SCE and HPRT mutation frequencies were observed to rise in noncytotoxic dosages of NB-UVB exposure. BB-UVB and NB-UVB both produced a significant increase in CPD formation and ROS formation (p < 0.05); however, higher dosages were required for NB-UVB. These results suggest that NB-UVB is less cytotoxic and genotoxic than BB-UVB, but can still produce genotoxic effects even at noncytotoxic doses.

The mutagenesis moonshot: The propitious beginnings of the environmental mutagenesis and genomics society.

A mutagenesis moonshot addressing the influence of the environment on our genetic wellbeing was launched just 2 months before astronauts landed on the moon. Its impetus included the discovery that X-rays (Muller HJ. [1927]: Science 64:84-87) and chemicals (Auerbach and Robson. [1946]: Nature 157:302) were germ-cell mutagens, the introduction of a growing number of untested chemicals into the environment after World War II, and an increasing awareness of the role of environmental pollution on human health. Due to mounting concern from influential scientists that germ-cell mutagens might be ubiquitous in the environment, Alexander Hollaender and colleagues founded in 1969 the Environmental Mutagen Society (EMS), now the Environmental Mutagenesis and Genomics Society (EMGS); Frits Sobels founded the European EMS in 1970. As Fred de Serres noted, such societies were necessary because protecting populations from environmental mutagens could not be addressed by existing scientific societies, and new multidisciplinary alliances were required to spearhead this movement. The nascent EMS gathered policy makers and scientists from government, industry, and academia who became advocates for laws requiring genetic toxicity testing of pesticides and drugs and helped implement those laws. They created an electronic database of the mutagenesis literature; established a peer-reviewed journal; promoted basic and applied research in DNA repair and mutagenesis; and established training programs that expanded the science worldwide. Despite these successes, one objective remains unfulfilled: identification of human germ-cell mutagens. After 50 years, the voyage continues, and a vibrant EMGS is needed to bring the mission to its intended target of protecting populations from genetic hazards. Environ. Mol. Mutagen. 61:8-24, 2020. © 2019 Wiley Periodicals, Inc.

Mutagenic Effects of a 2-Deoxyribonolactone-Thymine Glycol Tandem DNA Lesion in Human Cells.

Tandem DNA lesions containing two contiguously damaged nucleotides are commonly formed by ionizing radiation. Their effects on replication in mammalian cells are largely unknown. Replication of isolated 2-deoxyribonolactone (L), thymine glycol (Tg), and tandem lesion 5'-LTg was examined in human cells. Although nearly 100% of Tg was bypassed in HEK 293T cells, L was a significant replication block. 5'-LTg was an even stronger replication block with 5% TLS efficiency. The mutation frequency (MF) of Tg was 3.4%, which increased to 3.9% and 4.8% in pol ι- and pol κ-deficient cells, respectively. An even greater increase in the MF of Tg (to ∼5.5%) was observed in cells deficient in both pol κ and pol ζ, suggesting that they work together to bypass Tg in an error-free manner. Isolated L bypass generated 12-18% one-base deletions, which increased as much as 60% in TLS polymerase-deficient cells. The fraction of deletion products also increased in TLS polymerase-deficient cells upon 5'-LTg bypass. In full-length products and in all cell types, dA was preferentially incorporated opposite an isolated L as well as when it was part of a tandem lesion. However, misincorporation opposite Tg increased significantly when it was part of a tandem lesion. In wild type cells, targeted mutations increased about 3-fold to 9.7% and to 17.4, 15.9, and 28.8% in pol κ-, pol ζ-, and pol ι-deficient cells, respectively. Overall, Tg is significantly more miscoding as part of a tandem lesion, and error-free Tg replication in HEK 293T cells requires participation of the TLS polymerases.

Whole-exome sequencing reveals the impact of UVA light mutagenesis in xeroderma pigmentosum variant human cells.

UVA-induced mutagenesis was investigated in human pol eta-deficient (XP-V) cells through whole-exome sequencing. In UVA-irradiated cells, the increase in the mutation frequency in deficient cells included a remarkable contribution of C>T transitions, mainly at potential pyrimidine dimer sites. A strong contribution of C>A transversions, potentially due to oxidized bases, was also observed in non-irradiated XP-V cells, indicating that basal mutagenesis caused by oxidative stress may be related to internal tumours in XP-V patients. The low levels of mutations involving T induced by UVA indicate that pol eta is not responsible for correctly replicating T-containing pyrimidine dimers, a phenomenon known as the 'A-rule'. Moreover, the mutation signature profile of UVA-irradiated XP-V cells is highly similar to the human skin cancer profile, revealing how studies involving cells deficient in DNA damage processing may be useful to understand the mechanisms of environmentally induced carcinogenesis.

Asymmetric repair of UV damage in nucleosomes imposes a DNA strand polarity on somatic mutations in skin cancer.

Nucleosomes inhibit excision repair of DNA damage caused by ultraviolet (UV) light, and it has been generally assumed that repair inhibition is equivalent on both sides of the nucleosome dyad. Here, we use genome-wide repair data to show that repair of UV damage in nucleosomes is asymmetric. In yeast, nucleosomes inhibit nucleotide excision repair (NER) of the nontranscribed strand (NTS) of genes in an asymmetric manner, with faster repair of UV damage occurring on the 5' side of the nucleosomal DNA. Analysis of genomic repair data from UV-irradiated human cells indicates that NER activity along the NTS is also elevated on the 5' side of nucleosomes, consistent with the repair asymmetry observed in yeast nucleosomes. Among intergenic nucleosomes, repair activity is elevated on the 5' side of both DNA strands. The distribution of somatic mutations in nucleosomes shows the opposite asymmetry in NER-proficient skin cancers, but not in NER-deficient cancers, indicating that asymmetric repair of nucleosomal DNA imposes a strand polarity on UV mutagenesis. Somatic mutations are enriched on the relatively slow-repairing 3' side of the nucleosomal DNA, particularly at positions where the DNA minor groove faces away from the histone octamer. Asymmetric repair and mutagenesis are likely caused by differential accessibility of the nucleosomal DNA, a consequence of its left-handed wrapping around the histone octamer.

DNA Copy Number Variations as Markers of Mutagenic Impact.

DNA copy number variation (CNV) occurs due to deletion or duplication of DNA segments resulting in a different number of copies of a specific DNA-stretch on homologous chromosomes. Implications of CNVs in evolution and development of different diseases have been demonstrated although contribution of environmental factors, such as mutagens, in the origin of CNVs, is poorly understood. In this review, we summarize current knowledge about mutagen-induced CNVs in human, animal and plant cells. Differences in CNV frequencies induced by radiation and chemical mutagens, distribution of CNVs in the genome, as well as adaptive effects in plants, are discussed. Currently available information concerning impact of mutagens in induction of CNVs in germ cells is presented. Moreover, the potential of CNVs as a new endpoint in mutagenicity test-systems is discussed.

Unexpected behavior of DNA polymerase Mu opposite template 8-oxo-7,8-dihydro-2'-guanosine.

DNA double-strand breaks (DSBs) resulting from reactive oxygen species generated by exposure to UV and ionizing radiation are characterized by clusters of lesions near break sites. Such complex DSBs are repaired slowly, and their persistence can have severe consequences for human health. We have therefore probed DNA break repair containing a template 8-oxo-7,8-dihydro-2'-guanosine (8OG) by Family X Polymerase µ (Pol µ) in steady-state kinetics and cell-based assays. Pol µ tolerates 8OG-containing template DNA substrates, and the filled products can be subsequently ligated by DNA Ligase IV during Nonhomologous end-joining. Furthermore, Pol µ exhibits a strong preference for mutagenic bypass of 8OG by insertion of adenine. Crystal structures reveal that the template 8OG is accommodated in the Pol µ active site with none of the DNA substrate distortions observed for Family X siblings Pols ß or λ. Kinetic characterization of template 8OG bypass indicates that Pol µ inserts adenosine nucleotides with weak sugar selectivity and, given the high cellular concentration of ATP, likely performs its role in repair of complex 8OG-containing DSBs using ribonucleotides.