The far-reaching effects of mutagens on human health
IMAGE: MICHAEL LYNCH IS THE DIRECTOR OF THE BIODESIGN CENTER FOR MECHANISMS OF EVOLUTION AND PROFESSOR AT ASU'S SCHOOL OF LIFE SCIENCES. view more
CREDIT: THE BIODESIGN INSTITUTE AT ARIZONA STATE UNIVERSITY
In order to survive, flourish and successfully reproduce, organisms rely on a high degree of genetic stability. Mutagenic agents, which can threaten the integrity of the genetic code by causing mutations in DNA, pose a serious risk to human health. They have long been implicated in a range of genetically inherited afflictions, as well as cancer, aging and neurodegenerative diseases like Alzheimer's.
It now appears that mutagenic threats to a cell's subtle machinery may be far more widespread than previously appreciated. In a new study, Michael Lynch and his colleagues demonstrate that DNA mutation itself may represent only a fraction the health-related havoc caused by mutagens.
The study highlights the ability of mutagenic compounds to also affect the process of transcription, during which a DNA sequence is converted (or transcribed) to mRNA, an intermediary stage preceding translation into protein.
The research findings, (which highlight mutagenic transcription errors in yeast, worms, flies and mice), suggest that the harmful effects of mutagens on transcription are likely much more pervasive than previously appreciated--a fact that may have momentous implications for human health.
"Our results have the potential to completely transform the way we think about the consequences of environmental mutagens," Lynch says.
Professor Lynch is the director of the Biodesign Center for Mechanisms in Evolution and a researcher in ASU's School of Life Sciences.
The research results appear in the current issue of the journal PNAS.
Cells under threat
Due to their important role in disease processes, mutagenic compounds have long been a topic of intensive scientific study. Such agents include sunlight and other sources of radiation, chemotherapeutics, toxic byproducts of cellular metabolism, or chemicals present in food and water.
Mutagens can inflict damage to the DNA, which can later snowball when cells divide, and DNA replication multiplies these errors. Such mutations, if not corrected through DNA proofreading mechanisms, can be passed to subsequent generations and depending on the location at which they appear along the human DNA strand's three billion letter code may seriously impact health, in some cases, with lethal results.
But even if repaired prior to replication, transiently damaged DNA can also interfere with transcription--the process of producing RNA from a DNA sequence. This can happen when RNA polymerase, an enzyme that moves along a single strand of DNA, producing a complementary RNA strand, reads a mutated sequence of DNA, causing an error in the resulting RNA transcript.
Because RNA transcripts are the templates for producing proteins, transcription errors can produce aberrant proteins harmful to health or terminate protein synthesis altogether. It is already known that even under the best of conditions, transcript error rates are orders of magnitude higher than those at the DNA level.
RNA: a string of errors?
While the existence of transcription errors has long been recognized, their quantification has been challenging. The new study describes a clever technique for ferreting out transcription errors caused by mutagens and separating these from experimental artifacts--mutations caused during library preparation of RNA transcripts through processes of reverse-transcription and sequencing.
The method described involves the use of massively parallel sequencing technology to identify only those errors in RNA sequence directly caused by the activity of a mutagen. The results demonstrate that at least some mutagenic compounds are potent sources of both genomic mutations and abundant transcription errors.
The circular sequencing assay outlined in the study creates redundancies in the reverse-transcribed message, providing a means of proofreading the resultant linear DNA. In this way, researchers can confirm that the transcription errors observed are a result of the mutagen's effects on transcription and not an artifact of sample preparation.
The DNA molecule has been shown to be particularly vulnerable to a class of mutagens known as alkylating agents. One of these, known as MNNG, was used to inflict transcriptional errors on the four study organisms. The effects observed were dose-dependent, with higher levels of mutagen causing a corresponding increase in transcriptional errors.
Hidden mistakes may be costly to health
Transcription errors differ from mutations in the genome in at least one vital respect. While DNA replication during cell division acts to amplify mutations to the genome, transcription errors can accumulate in non-dividing cells, with a single mutated DNA template giving rise to multiple abnormal RNA transcripts.
The full effects of these transcription errors on human health remain largely speculative because they have not been amenable to study until now. Using the new technique, researchers can mine the transcriptome--the full library of a living cell's RNA transcripts, searching for errors caused by mutagens.
While the new research offers hope for a more thorough understanding of the relationship between various mutagens and human health, it is also a cautionary tale. A preoccupation with mutational defects in DNA sequence may have blinded science to the potential effects of agents that result in transcription errors without leaving permanent traces in the genome.
This fact raises the possibility that a broad range of environmental factors as well as chemicals and foods deemed safe for human consumption are in need of careful reevaluation based on their potential for producing transcriptional mutagenesis. Further, transcriptional errors in both dividing and non-dividing cell types are likely key players in the complex processes of physical aging
Beyond changing DNA itself, mutagens also cause errors in gene transcription
The discovery that toxic stressors can cause errors in gene transcription opens new avenues of research on diseases such as Alzheimer's and Parkinson's and sheds light on the potential role of the "transcriptome" in aging.
IMAGE: ASSISTANT PROFESSOR MARC VERMULST view more
CREDIT: USC/STEPHANIE KLEINMAN
Exposure to mutagens, or mutation-causing agents, can not only bring about changes in DNA but also appear to induce errors when genes are transcribed to make proteins, which may be an important factor in age-related diseases.
USC Leonard Davis School of Gerontology Assistant Professor Marc Vermulst and colleagues made the discovery by using state-of-the-art circle sequencing techniques to determine how frequently molecules called RNA polymerases make mistakes when they read (or "transcribe") our DNA. RNA polymerases transcribe DNA to make temporary copies of genes, which are then used to build all of the proteins required to keep us alive and healthy.
Transcription errors vastly outnumber DNA mutations
Vermulst compared our cells to a busy kitchen, teeming with hundreds of chefs that are all making dishes out of a single recipe book. Because it's so busy, they cannot take the recipe book with them when an order comes in. So instead they send the kitchen staff to the recipe book to read the recipes as carefully as possible and then bring the instructions to the chefs. Our cells work in a very similar manner. When an "order" for a protein comes in, RNA polymerases are sent to our genome (or in other words, our recipe book), to make a temporary copy of a gene. That temporary copy is then brought to the chefs, who cook the protein just like the message they received dictates. In this example, transcription errors could be an incorrect amount or ingredient that wasn't properly recorded by the person jotting down the recipe.
"The molecule doing the reading and writing is what's introducing the errors, even if the DNA itself isn't mutated," he explained.
To demonstrate that a mutagen - an agent that can cause a genetic mutation - can induce these errors, Vermulst and his team exposed yeast cells to the chemical N-Methyl-N?-nitro-N-nitrosoguanidine (MNNG), then screened for transcription errors. The cells exposed to MNNG displayed many more transcription errors than the unexposed cells, and in addition, the rate of transcription errors vastly outnumbered the rate of DNA mutations. The team confirmed similar results when the experiments were repeated in cells from the worm species C. elegans, fruit fly D. melanogaster and mice.
DNA mutations occur when the genome is inaccurately copied during cell division, leaving the newly formed cells with a mistake in their DNA. However, a few types of cells, including neurons and muscle cells, rarely divide in adults. These cells all still need to transcribe proteins, which means that harmful errors within these cells are much more likely to arise from transcription, Vermulst explained.
"There are a hundredfold more transcription errors being made for every DNA mutation that eventually arises," he said.
A possible role in several diseases
The genes that code for a protein not only instruct which amino acids to put in what order but also control the specific shape into which the finished protein folds itself. Transcription errors often cause proteins to misfold into a dysfunctional shape, which can result in clusters, or plaques, of nonfunctioning proteins that hinder healthy cell function. This raises questions of how these errors may play roles in diseases such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS) and others, Vermulst said.
In future research, Vermulst is pushing for more investigation into whether other substances known to cause DNA mutations also affect transcription, as well as if there are any substances previously thought of as safe that may be in fact inducing transcription errors.
"This is potentially a really important finding in the context of genetic toxicology: a new mechanism by which all these molecules - from exposures in our environment or from our lifestyle choices - can result in pathology," he said. "There could potentially be molecules that we're eating and drinking that are deemed safe because they don't result in any genetic changes, but do result in transcription errors, that have gone completely unnoticed because nobody had a tool to see whether or not that was happening."
He also hopes that the research will make new links between established pillars of aging research - DNA damage, mitochondrial dysfunction, oxidative species and others - and connect them in a mechanistic way to detrimental outcomes such as Alzheimer's, Parkinson's and cancer. It may also help identify sources of the symptoms in DNA repair deficiency disorder, in which patients are unable to repair damage to their genome properly and often results in accelerated aging or increased cancer risk.
While recent years have seen increased interest in the "transcriptome" - the entirety of what is transcribed from a genome - Vermulst wants to focus on the accuracy of what's being transcribed and not just the amount of each protein produced. He hopes this quality-over-quantity approach offers new insight into the fundamental processes of diseases.
"If you've done the same thing a hundred times and you don't get a solution for your problem, it might be something that you've overlooked," he said. "So we're trying to find this something else."
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Vermulst's co-corresponding author for the study was Michael Lynch of Arizona State University, and first author was Clark Fritsch of the University of Pennsylvania. Other coauthors included Berenice Benayoun, Prakroothi S. Danthi, Eric McGann, Jessica LaGosh and Claire Chung of the USC Leonard Davis School; Jean-Francois Gout of Mississippi State University; Suraiya Haroon, Atif Towheed, Yuanquan Song and Douglas Wallace of the Children's Hospital of Philadelphia; Xinmin Zhang of BioInfoRx, Inc.; and Stephen Simpson and Kelley Thomas of the University of New Hampshire.
The study, "Genome-wide surveillance of transcription errors in response to genotoxic stress," appeared online in Proceedings of the National Academy of Sciences on December 21, 2020. This research was supported by the National Institute on Aging Award R01AG054641 and American Federation for Aging Research young investigator award in Alzheimer's disease to Vermulst; the Multidisciplinary University Research Initiative Awards W911NF-09-1-0444 from the US Army Research Office and NIH Award R35-GM122566-01 to Lynch; and Environmental Toxicology Training Grant T32ES019851 by the National Institute of Environmental Health Sciences to Fritsch.
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