Sequence-Specific Quantitation of Mutagenic DNA Damage via Polymerase Amplification with an Artificial Nucleotide.

DNA mutations can result from replication errors due to different forms of DNA damage, including low-abundance DNA adducts induced by reactions with electrophiles. The lack of strategies to measure DNA adducts within genomic loci, however, limits our understanding of chemical mutagenesis. The use of artificial nucleotides incorporated opposite DNA adducts by engineered DNA polymerases offers a potential basis for site-specific detection of DNA adducts, but the availability of effective artificial nucleotides that insert opposite DNA adducts is extremely limited, and furthermore, there has been no report of a quantitative strategy for determining how much DNA alkylation occurs in a sequence of interest. In this work, we synthesized an artificial nucleotide triphosphate that is selectively inserted opposite O6-carboxymethyl-guanine DNA by an engineered polymerase and is required for DNA synthesis past the adduct. We characterized the mechanism of this enzymatic process and demonstrated that the artificial nucleotide is a marker for the presence and location in the genome of O6-carboxymethyl-guanine. Finally, we established a mass spectrometric method for quantifying the incorporated artificial nucleotide and obtained a linear relationship with the amount of O6-carboxymethyl-guanine in the target sequence. In this work, we present a strategy to identify, locate, and quantify a mutagenic DNA adduct, advancing tools for linking DNA alkylation to mutagenesis and for detecting DNA adducts in genes as potential diagnostic biomarkers for cancer prevention.

Immunological and mass spectrometry-based approaches to determine thresholds of the mutagenic DNA adduct O6-methylguanine in vivo.

N-nitroso compounds are alkylating agents, which are widespread in our diet and the environment. They induce DNA alkylation adducts such as O6-methylguanine (O6-MeG), which is repaired by O6-methylguanine-DNA methyltransferase (MGMT). Persistent O6-MeG lesions have detrimental biological consequences like mutagenicity and cytotoxicity. Due to its pivotal role in the etiology of cancer and in cytotoxic cancer therapy, it is important to detect and quantify O6-MeG in biological specimens in a sensitive and accurate manner. Here, we used immunological approaches and established an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to monitor O6-MeG adducts. First, colorectal cancer (CRC) cells were treated with the methylating anticancer drug temozolomide (TMZ). Immunofluorescence microscopy and an immuno-slot blot assay, both based on an adduct-specific antibody, allowed for the semi-quantitative, dose-dependent assessment of O6-MeG in CRC cells. Using the highly sensitive and specific UPLC-MS/MS, TMZ-induced O6-MeG adducts were quantified in CRC cells and even in peripheral blood mononuclear cells exposed to clinically relevant TMZ doses. Furthermore, all methodologies were used to detect O6-MeG in wildtype (WT) and MGMT-deficient mice challenged with the carcinogen azoxymethane. UPLC-MS/MS measurements and dose-response modeling revealed a non-linear formation of hepatic and colonic O6-MeG adducts in WT, whereas linear O6-MeG formation without a threshold was observed in MGMT-deficient mice. Collectively, the UPLC-MS/MS analysis is highly sensitive and specific for O6-MeG, thereby allowing for the first time for the determination of a genotoxic threshold upon exposure to O6-methylating agents. We envision that this method will be instrumental to monitor the efficacy of methylating chemotherapy and to assess dietary exposures.

Regulation of human MutYH DNA glycosylase by the E3 ubiquitin ligase mule.

Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.

Correction to "Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures".

[This corrects the article DOI: 10.1021/acscentsci.2c01100.].

Quantification and Mapping of Alkylation in the Human Genome Reveal Single Nucleotide Resolution Precursors of Mutational Signatures.

Chemical modifications to DNA bases, including DNA adducts arising from reactions with electrophilic chemicals, are well-known to impact cell growth, miscode during replication, and influence disease etiology. However, knowledge of how genomic sequences and structures influence the accumulation of alkylated DNA bases is not broadly characterized with high resolution, nor have these patterns been linked with overall quantities of modified bases in the genome. For benzo(a) pyrene (BaP), a ubiquitous environmental carcinogen, we developed a single-nucleotide resolution damage sequencing method to map in a human lung cell line the main mutagenic adduct arising from BaP. Furthermore, we combined this analysis with quantitative mass spectrometry to evaluate the dose-response profile of adduct formation. By comparing damage abundance with DNase hypersensitive sites, transcription levels, and other genome annotation data, we found that although overall adduct levels rose with increasing chemical exposure concentration, genomic distribution patterns consistently correlated with chromatin state and transcriptional status. Moreover, due to the single nucleotide resolution characteristics of this DNA damage map, we could determine preferred DNA triad sequence contexts for alkylation accumulation, revealing a characteristic DNA damage signature. This new BaP damage signature had a profile highly similar to mutational signatures identified previously in lung cancer genomes from smokers. Thus, these data provide insight on how genomic features shape the accumulation of alkylation products in the genome and predictive strategies for linking single-nucleotide resolution in vitro damage maps with human cancer mutations.

Effects of attention shifts to stationary objects during steady-state smooth pursuit eye movements.

A number of studies have shown that stationary backgrounds compromise smooth pursuit eye movements. It has been suggested that poor attentional selection of the pursuit target was responsible for reductions of pursuit gain. To quantify the detrimental effects of attention, we instructed observers to either pay attention to background objects or to ignore them. The to-be-attended object was indicated by peripheral or central cues. Strong reductions of pursuit gain occurred when the following conditions were met: (a) the subject payed attention to the object (b) a salient event was present, for instance the onset of the target or cue and (c) the attended target produced retinal motion. Removing any of the three conditions resulted in no or far smaller decreases of pursuit gain. Further, decreases in pursuit gain were present with perceptual discrimination and simple manual detection.

Iron phosphate nanoparticles for food fortification: Biological effects in rats and human cell lines.

Nanotechnology offers new opportunities for providing health benefits in foods. Food fortification with iron phosphate nanoparticles (FePO4 NPs) is a promising new approach to reducing iron deficiency because FePO4 NPs combine high bioavailability with superior sensory performance in difficult to fortify foods. However, their safety remains largely untested. We fed rats for 90 days diets containing FePO4 NPs at doses at which iron sulfate (FeSO4), a commonly used food fortificant, has been shown to induce adverse effects. Feeding did not result in signs of toxicity, including oxidative stress, organ damage, excess iron accumulation in organs or histological changes. These safety data were corroborated by evidence that NPs were taken up by human gastrointestinal cell lines without reducing cell viability or inducing oxidative stress. Our findings suggest FePO4 NPs appear to be as safe for ingestion as FeSO4.

Mislocalization of flashes during smooth pursuit hardly depends on the lighting conditions.

Targets that are briefly flashed during smooth pursuit eye movements are mislocalized in the direction of motion (forward shift) and away from the fovea (spatial expansion). Hansen [Hansen, R. M. (1979). Spatial localization during pursuit eye movements. Vision Research 19(11), 1213-1221] reported that these errors are not present for fast motor responses in the dark, whereas Rotman et al. [Rotman, G., Brenner, E., Smeets, J. B. (2004). Quickly tapping targets that are flashed during smooth pursuit reveals perceptual mislocalizations. Experimental Brain Research 156(4), 409-414] reported that they are present for fast motor responses in the light. To evaluate whether the lighting conditions are the critical factor, we asked observers to point to the positions of flashed objects during smooth pursuit either in the dark or with the room lights on. In a first experiment, the flash, which could appear at 1 of 15 different positions, was always shown when the eye had reached a certain spatial position. We found a forward bias and spatial expansion that were independent of the target and ambient luminance. In a second experiment, the flash was always shown at the same retinal position, but the spatial position of the eye at the moment of flash presentation was varied. In this case we found differences between the luminance conditions, in terms of how the errors depended on the velocity and position on the trajectory. We also found specific conditions in which people did not mislocalize the target in the direction of pursuit at all. These findings may account for the above-mentioned discrepancy. We conclude that although the lighting conditions do influence the localization errors under some circumstances, it is certainly not so that such errors are absent whenever the experiment is conducted in the dark.

Impact of ribonucleotide incorporation by DNA polymerases ß and λ on oxidative base excision repair.

Oxidative stress is a very frequent source of DNA damage. Many cellular DNA polymerases (Pols) can incorporate ribonucleotides (rNMPs) during DNA synthesis. However, whether oxidative stress-triggered DNA repair synthesis contributes to genomic rNMPs incorporation is so far not fully understood. Human specialized Pols ß and λ are the important enzymes involved in the oxidative stress tolerance, acting both in base excision repair and in translesion synthesis past the very frequent oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxo-G). We found that Pol ß, to a greater extent than Pol λ can incorporate rNMPs opposite normal bases or 8-oxo-G, and with a different fidelity. Further, the incorporation of rNMPs opposite 8-oxo-G delays repair by DNA glycosylases. Studies in Pol ß- and λ-deficient cell extracts suggest that Pol ß levels can greatly affect rNMP incorporation opposite oxidative DNA lesions.

Visual short-term memory during smooth pursuit eye movements.

Visual short-term memory (VSTM) was probed while observers performed smooth pursuit eye movements. Smooth pursuit keeps a moving object stabilized in the fovea. VSTM capacity for position was reduced during smooth pursuit compared with a condition with eye fixation. There was no difference between a condition in which the items were approximately stabilized on the retina because they moved with the pursuit target and a condition in which the items moved across the retina because they were stationary in space. The reduction of capacity for position was eliminated when miniature items were presented on the pursuit target. Similarly, VSTM capacity for color did not differ between smooth pursuit and fixation. The results suggest that visuospatial attention is tied to the target during smooth pursuit, which impairs VSTM for the position of peripheral objects. Sensory memory during smooth pursuit was only slightly impaired.

Training in reproductive endocrinology and infertility and assisted reproductive technologies: options and worldwide needs.

Standardized, high-quality training in reproductive endocrinology, infertility, and assisted reproductive technologies (REI-ART) faces challenges owing to the high-tech nature of ART and the important country-to-country differences in clinical practice and regulations overseeing training. Moreover, while the training capacity of the classical by-fellowship training platforms is shrinking, an increasing demand for REI-ART specialists is coming from emerging countries. To meet this expanding need for REI-ART specialists, we propose a novel by-network model linking a reference training center to satellite practical training sites. Simulation should be used more extensively to achieve competency before initiating live clinical experience, analogous to the highly effective training systems that have been used in aviation for decades. Large ART databases that exist because of obligations to report ART activity and results constitute unique yet so far untapped sources for developing by-scenario simulation training models. Online training materials incorporating these state-of-the-art information technology tools could be developed as a means of fulfilling training needs worldwide.

Could safety boards provide a valuable tool to enhance the safety of reproductive medicine?

Medicine and aviation have a striking number of similarities. Both are led by highly-trained individuals performing complex tasks that are critical to outcomes. They also integrate the efforts of other professionals to assure that the flight, procedures, or processes are completed successfully. Also in common, is the potential for errors to have catastrophic and even life-threatening consequences. Both aviation and medicine have responded to this complex operating environment by building safety programs. Unfortunately, those in medicine have not been optimal in reducing significant adverse outcomes, including deaths. It has been suggested that given the parallels, that aviation safety programs might be adapted to clinical medicine. One such measure would the formation of a Clinical Safety Board (CSB) modeled after the National Transportation Safety Board (NTSB). Such a board would collect data across the nation and determine root causes of errors. They may then provide recommendations to professional societies and regulatory agencies for consideration for implementation. Such programs would be dependent on accurate and thorough reporting. Indemnification, similar to that enacted by the federal government for aviation, would be critical. In the end, a CSB should empower better patient care with reduced liability to the providers and programs.