We analyzed mutations arising in cancer cells in nine Protein Arginine Methyltransferases (PRMT1–9) reported on the Catalogue of Somatic Mutations in Cancer (COSMIC). Most mutations are single amino acid substitutions. Consistent with this interpretation, and as previously reported, most PRMT genes are over-expressed in cancer cells. We used the CHASMPlus and VEST4 algorithms to classify mutations as either driver (probability of causing cancer) or pathogenic (probability of causing any disease), respectively. Driver mutations are virtually non-existent in any of the PRMT genes, which was not unexpected because none of the genes are classified as a driver by OncoGenomics. Pathogenic mutations were identified in six genes. We used in silico structural analysis to analyze high-frequency mutations and found that PRMT6 mutations strongly affected protein structure, whereas PRMT1 mutations tended to not impact the structure. Mutation co-occurrence between the PRMT genes is likely except between PRMT5 and PRMT6 suggesting that cells can largely tolerate mutations in more than one PRMT. Although the study presented here uses in silico analysis to catalogue PRMT1–9 mutations, it highlights interesting trends that may require further experimental analysis.
Deuterium (D) alters biochemical reaction kinetics through the hydrogen/deuterium kinetic isotope effect (KIE), but its role in spontaneous mutagenesis remains controversial. Here, we investigated the effects of deuterium on mutagenesis in Escherichia coli strains AB1157 and BMH71-18 under either deuterium-labeled medium (D-broth) or unlabeled medium (H-broth) conditions. The growth rates of both strains in D-broth were reduced by 2.77and 3.90-fold, respectively, compared with those in H-broth. Stationary-phase viability was not significantly affected in either strain, indicating that deuterium inhibits growth kinetically rather than through cytotoxicity. Spontaneous mutation frequency was evaluated using rifampicin resistance (Rif(R)). A significant 5.57-fold increase in RifR mutant frequency was observed in BMH71-18 under deuterated conditions, whereas no significant increase was detected in AB1157. Whole-genome sequencing was therefore focused on BMH71-18, the strain showing the clearest mutational response to deuteration. Whole-genome sequencing of randomly selected colonies revealed a 5.35-fold increase in mutation events under deuterated conditions. Single-base substitutions predominated in both conditions, with transition mutations accounting for more than 90% of all substitutions. In contrast, no significant differences were detected in transversions, small insertions, deletions, or structural variants. These findings are consistent with the possibility that deuterium promotes mutation fixation by prolonging the persistence of rare tautomeric states during DNA replication. Collectively, our results suggest that deuterium enhances mutation fixation in E. coli rather than acting as a primary mutagen and is associated with a strongly transition-biased mutation spectrum.
APOBEC3A is a single-stranded DNA cytidine deaminase that is involved in mutagenic processes in various cancers. Ample evidence indicates that APOBEC3A causes two major mutational signatures (SBS2 and SBS13) in cancer cells and cellular model systems, but the detailed molecular mechanisms that generate these two signatures from a single deaminase remain unclear. APOBEC3A-induced mutations are preferentially observed at DNA hairpin structures and are often observed as clusters in cancer genomes, but the molecular mechanisms behind the clustered mutations are unclear. Here, we developed a cell-free system to analyze the mutations on APOBEC3A-treated DNA and successfully reproduced SBS2 and a portion of SBS13 by using specific combinations of DNA polymerases. While SBS2 can be produced by a replicative DNA polymerase, SBS13 requires the activities of uracil DNA glycosylase, Rev1, and Pol zeta. We also found that the DNA hairpin structures have positive and negative influences on deaminase activity depending on the position of cytosine residues within the loop. In our system, APOBEC3A-induced mutagenesis showed positive cooperativity by which one mutation stimulated a second mutation at up to 50-nt distance. This cooperative effect promoted clustered mutagenesis in vitro, but little continuity of mutations was observed among the multi-mutation products. These observations support an aggregation-mediated mechanism of clustered mutagenesis.
OECD TG 489 recognizes that some modifications of the in vivo comet assay (e.g., longer electrophoresis times) can detect DNA crosslink inductions expressed as DNA migration decreases. But the application of these methods in regulated safety testing has been limited because identifying an in vivo crosslinking positive control for every tissue that can be evaluated with the comet assay can be very challenging. Most crosslinking agents induce crosslinks in just one tissue (e.g., site of contact) with the agent’s toxicity limiting exposure and effects induced in other tissues. This complication can require different positive control compounds with additional animals for each compound to adequately evaluate crosslinks in the multiple tissues typically evaluated with the comet assay. Our objective was to develop a method for generating concurrent in vivo crosslinking positive control comet data for any tissue without using additional animals. We accomplished this by preparing for each tissue evaluated extra comet slides from the conventional positive control (EMS) dosed animals and ex vivo crosslinking the heavily damaged cells on these slides with 10% neutral buffered formalin (NBF) immediately before lysis. These ex vivo crosslinked slides were then electrophoresed with the rest of the study slides with the same standard conditions and electrophoresis time. The conventional comet study slides prepared from the liver, duodenum, glandular stomach, and lung tissues collected from the EMS dosed animals and lysed without ex vivo crosslinking expressed a statistically significant DNA migration increase when compared to the concurrent vehicle control dose group. The ex vivo crosslinked slides from the EMS dose group tissue samples expressed a statistically significant decrease in DNA migration when compared to the concurrent vehicle control group. Although no statistical analysis was conducted, it is clear by the data that the ex vivo crosslinked slides from the EMS dose group tissue samples also expressed a significant decrease compared to the same samples from the EMS dose group without the ex vivo NBF treatment. This data demonstrate that the method can be easily incorporated into any existing comet study design to evaluate crosslink induction in any comet assay tissue with minimal effort or cost and without the use of any additional animals.
Background Antibiotic resistance in Helicobacter pylori poses a significant challenge to the effective eradication of infection worldwide. Understanding molecular mechanisms of resistance is essential for guiding treatment strategies. This study aimed to investigate the molecular basis of antimicrobial resistance in Helicobacter pylori isolates and their associated mutation frequencies. Methods In this cross-sectional study, gastric biopsy specimens were collected from 203 patients at Rizgary Hospital in Erbil, Kurdistan Region, Iraq, who underwent endoscopy for dyspepsia-related symptoms. Of the 137 positive patients, 91 Helicobacter pylori isolates were confirmed by colony morphology, Gram staining, and biochemical tests; 63 were successfully subcultured for antimicrobial susceptibility testing (culture success rate: 69.2%). Antimicrobial susceptibility testing was performed by the agar dilution method to determine the minimum inhibitory concentrations. The sequences of specific genes were examined and analysed by next-generation sequencing. Multiple sequence comparisons were performed to identify resistance-related genes and mutations, using 26695 (NC_000915.1) as the reference genome. Results Only two isolates (3.17%) were susceptible to all antibiotics examined. The frequency of metronidazole resistance was highest (85.71%), followed by levofloxacin (55.55%), clarithromycin (52.38%), amoxicillin (26.98%), tetracycline (6.35%), and rifabutin (4.76%). Mutations in the rdxA and frxA genes correlated with metronidazole resistance, while GyrA protein mutations at positions 87 and 91 were linked to levofloxacin resistance. Clarithromycin resistance was mainly associated with A2142G and A2143G mutations in 23S rRNA. Amoxicillin resistance (26.98%) was associated with mutations in the pbp1A gene, whereas resistance to tetracycline and rifabutin was infrequent. Conclusions: This study provides the first molecular surveillance data on antimicrobial resistance in Helicobacter pylori in northern Iraq, offering valuable regional evidence to guide local eradication strategies. The relatively high amoxicillin resistance, together with the elevated resistance to metronidazole, levofloxacin, and clarithromycin, underscores the need for susceptibility-guided therapy and continuous local antimicrobial resistance surveillance.
