A recent study has challenged a long-held belief regarding the origin of the most prevalent DNA mutation observed in our genomes, which is implicated in various genetic disorders, including cancer.
A Ludwig Cancer Research study has challenged a long-held belief regarding the origin of the most prevalent DNA mutation observed in our genomes – one that is involved in several genetic disorders, including cancer.
Conducted by Ludwig Oxford Leadership Fellow Marketa Tomkova, along with postdoc Michael McClellan, Assistant Member Benjamin Schuster-Böckler, and Associate Investigator Skirmantas Kriaucionis, this study has significant implications for fundamental cancer biology as well as for evaluating carcinogenic risks related to environmental factors and our understanding of how drug resistance develops during cancer treatment. The results are published in the latest issue of Nature Genetics.
The specific mutation under investigation involves the incorrect conversion of cytosine (C)—one of the four DNA bases that encode our genes—into thymine (T). It was previously believed that this process primarily occurred due to a spontaneous chemical reaction with water. This reaction, known as deamination, is approximately twice as likely to occur when a cytosine is chemically marked by a methyl group, resulting in 5-methylcytosine, especially at locations in the DNA known as “CpG” sites, where cytosine is followed by guanine (G). This type of methylation is widespread throughout the genome and is crucial for regulating gene expression, which is vital for many cellular functions from embryonic development onward.
“It has been widely believed that C to T mutations arise from random chemical reactions,” said Tomkova. “Our research indicates that this view is not entirely accurate. In fact, these mutations are predominantly produced when a cell duplicates its genome, primarily due to the tendency of a crucial part of the cell’s DNA copying machinery to make errors when it encounters methylated cytosines.”
The Ludwig Oxford team first suspected a different mechanism a few years ago while analyzing cancer genome sequences shared by laboratories in the UK and Canada. They discovered that cancer cells with specific genetic anomalies were significantly more likely to exhibit CpG to TpG mutations.
These mutations were found in cells that struggled with repairing mismatched DNA due to errors produced during replication, particularly those harboring mutations in their DNA replication machinery, specifically in DNA polymerase ε (Pol ε), which normally proofreads new DNA strands and corrects errors. Both of these defects hinder DNA repair during cell division and are known to correlate with highly mutated tumors in cancer patients.
“This study would not have been feasible without the open sharing of data among researchers globally. We first identified a unique pattern at methylcytosine sites in the data we received from those laboratories and used public datasets to refine our hypothesis before conducting experimental tests,” commented Schuster-Böckler.
To validate their hypothesis, the researchers developed a novel and highly sensitive DNA sequencing method capable of distinguishing authentic errors made by Pol ε during DNA replication from experimental artifacts. They used their technique, called Polymerase Error Rate Sequencing (PER-seq), to analyze over 28 billion bases across more than 130 million DNA molecules, assessing the accuracy of both normal human Pol ε and the most common cancer-related variant of the enzyme.
Their findings indicated that the mutant Pol ε produced CpG to TpG mutations at rates comparable to those observed in cancer cells carrying that mutation. Even normal Pol ε created mutations at methylcytosine sites at seven times the rate of non-methylated cytosines.
These results, which directly connect the occurrence of CpG to TpG mutations to the process of cell division, clarify why these mutations tend to accumulate as organisms age. They also shed light on the variability of these mutations across different tissues and tumors due to the differing proliferation rates of various normal and cancerous cells.
“This means that tracking the accumulation of CpG to TpG mutations can serve as a biological clock to ascertain cellular age, which may be beneficial for research into how quickly different cancers grow before developing resistance to specific treatments,” stated Kriaucionis.
Moreover, the techniques developed from this research hold promise for cancer prevention. Understanding the proportion of mutations caused by errors during normal processes, like cell division, in relevant tissues is essential for accurately assessing how various environmental factors—such as chemical pollutants—can lead to cancer-causing DNA mutations.
This study received support from Ludwig Cancer Research, the Biotechnology and Biological Sciences Research Council, the Wellcome Trust, the National Institute for Health Research, the Conrad N. Hilton Foundation, and the Medical Research Council.
