It has often been a theme of my blog posts how, if we regard cells as the result of conscious (I won't use the term 'intelligent' as that's singularly inappropriate, as we shall see) design then the picture quickly emerges of a bungling incompetent, cobbling together Heath-Robinson solutions to problems of its own incompetent making and lack of foresight.
The entire system of epigenetics, for example, is only necessary in a multicellular organism with its cell specialisation because specialised cells only need a small subset of the entire genome, yet, because cells replicate using exactly the same process that single-celled organisms use, where the entire genome needs to be replicated in every daughter cell, the cells of multicellular organisms such as humans each receive the entire species genome. So, most of it needs to be turned off.
This is where the epigenetic system comes in where methyl groups are attached to key bases in the DNA which prevents that section being transcribed into RNA, effectively switching the gene off. There are other components to the epigenetic system, but that is the pertinent component as far as this particular blunder is concerned.
The problem starts when a methyl group is attached to a cytosine base (C in the CGTA genetic code) which is next to a guanine(G) base.
What do you know about C>T mutations at CpG dinucleotides in the human genome, what causes them and the problems they cause? C>T mutations at CpG dinucleotides are one of the most common point mutations in the human genome. These mutations occur due to a biochemical process involving cytosine in DNA. Here's an overview:It was thought that this sometimes causes a reaction with a molecule of water which results in the cytosine being chemically converted into a thymine base (T) to give a mutation in whatever gene this happens to take place. And some of these mutations go on to cause a cancer to develop.
Causes:
- Methylation of Cytosine: CpG dinucleotides (where a cytosine is followed by a guanine) are often methylated at the cytosine base, producing 5-methylcytosine. This methylation is part of an epigenetic mechanism that regulates gene expression.
- Deamination: When 5-methylcytosine undergoes spontaneous deamination, it is converted into thymine (T). This results in a C>T transition. Unlike unmethylated cytosine, which deaminates into uracil (easily recognized as foreign and repaired by the cell), the cytosine in CpG sites that deaminates to thymine leads to a permanent mutation because thymine is a natural DNA base and not flagged for repair.
Consequences and Problems:
- High Mutation Rate: CpG sites are mutation hotspots in the human genome because the methylated cytosine is much more prone to deamination. As a result, CpG dinucleotides are underrepresented in the human genome, as many have mutated over evolutionary time.
- Human Diseases: These C>T mutations can have significant biological consequences, especially in regulatory regions or coding sequences:
- Cancer: Many C>T mutations at CpG sites are associated with cancer, especially in tumor suppressor genes (e.g., the **p53** gene, where CpG mutations are common). The loss of gene function due to these mutations can drive oncogenesis.
- Genetic Disorders: Mutations at CpG sites can cause inherited diseases. For example, mutations in the gene responsible for cystic fibrosis and various neurological disorders can arise from CpG mutations.
- Evolutionary Impact: Over time, C>T transitions have reshaped the human genome, reducing the frequency of CpG dinucleotides. This is why they are rarer than expected based on random base pair distribution.
In summary, C>T mutations at CpG dinucleotides are caused by methylation and subsequent deamination of cytosine. These mutations can result in various diseases, particularly cancer, due to the high mutation rates at these sites.
But a team of researchers at Oxford University's Ludwig Cancer Research Centre have discovered that this is not entirely true. It's not a reaction with a water molecule that cause the mutation but rather another badly designed process - the error-prone, unintelligently designed replication process when a cell divides.
The team have just published their findings, open access, in Nature Genetics They have also described their research in Ludwig Cancer Research new release:
The cause of the genome’s commonest mutation has been reassigned
A Ludwig Cancer Research study has punctured a longstanding assumption about the source of the most common type of DNA mutation seen in the genome — one that contributes to many genetic diseases, including cancer.
Led by Ludwig Oxford Leadership Fellow Marketa Tomkova, postdoc Michael McClellan, Assistant Member Benjamin Schuster-Böckler and Associate Investigator Skirmantas Kriaucionis, the study has implications not only for basic cancer biology but also for such things as assessments of carcinogenic risk associated with environmental factors and our understanding of the emergence of drug resistance during cancer therapy. Its findings are reported in the current issue of Nature Genetics.
The mutation in question — in which cytosine (C), one of the four bases of DNA that spell out our genes, is erroneously switched to thymine (T) — was thought to be primarily the result of a spontaneous chemical reaction with water. This reaction, deamination, is about twice as likely to happen when a cytosine is chemically tagged by the addition of a molecule known as a methyl group to create 5-methylcytosine, which occurs in DNA at so-called “CpG” positions, where C is followed by the base guanine (G). Such tagging, commonly seen across the genome, plays a fundamental role in controlling the expression of genes and is therefore essential to pretty much every aspect of appropriate cellular function from embryonic development onward.
It has long been assumed that C to T mutations are caused by a random chemical reaction. Our study demonstrates that this is not entirely correct. Rather, the mutation is primarily produced when the cell copies its genome to divide and is mainly caused by the tendency of a key component of the cell’s DNA-copying machinery to make editing mistakes when it encounters methylated cytosines.
Marketa Tomkova, co-first author.
Ludwig Institute for Cancer Research
University of Oxford, Oxford, UK.
The Ludwig Oxford team got their first inkling of what was going on several years ago, when they examined sequences of cancer genomes shared with them by laboratories in the UK and Canada. They noticed in these data that cancer cells with certain genetic aberrations were far and away more likely to have CpG to TpG mutations.
These were cells known to be deficient in their ability to repair mismatched DNA sequences generated by mutations and those that bore mutations to a component of their DNA replication machinery, DNA polymerase ε (Pol ε), that proof-reads new DNA strands and edits out such errors. Both these defects interfere with DNA repair during cell division, and both are known to be associated with highly mutated tumors in cancer patients.
Our study would not have been possible without the free and open sharing of data between researchers around the world: we first spotted a peculiar pattern at methylcytosine sites in the data we received from those laboratories, and we used public data to refine our hypothesis before we even began to think of experimentally testing.
Benjamin Schuster-Böckler, co-senior author
Ludwig Institute for Cancer Research
University of Oxford, Oxford, UK.
To test their hypothesis, the researchers developed a new and very sensitive DNA sequencing technology that could discern genuine errors made by Pol ε during DNA replication from experimental artifacts. They applied their technique, dubbed Polymerase Error Rate Sequencing (PER-seq), to sequence over 28 billion bases across more than 130 million DNA molecules, measuring the accuracy of both normal human Pol ε and the most common cancer-associated mutant of the enzyme.
Their studies revealed that the mutant Pol ε produced CpG to TpG mutations at rates similar to those seen in cancer cells that carry that mutant. Even the normal Pol ε produced mutations at methylcytosine sites at seven times the rate observed for nonmethylated cytosines.
These findings, which directly link the incidence of CpG to TpG mutations to cell division, explain why these mutations tend to accumulate with age. They also explain why the mutation varies so much in frequency across tissues and tumors: because different types of both normal and cancerous cells proliferate at very different rates.
This also means that the accumulation of CpG to TpG mutations can be used like a clock to determine the age of cells, which could be useful to studies exploring, for example, how fast different cancers grow before acquiring resistance to different treatments.
Skirmantas Kriaucionis, co-senior author
Ludwig Institute for Cancer Research
University of Oxford, Oxford, UK.
Further, the methods developed for this study also have implications for cancer prevention. To accurately gauge how likely various environmental factors—such as chemical pollutants—are to induce cancer-causing DNA mutations, it helps to know what proportion of those mutations is caused by errors during normal processes, such as cell division, in relevant tissues.
AbstractOne consequence of this common form of mutation caused by an error-prone replication process is that the occurrence of C-G base pairs in the genome have tended to reduce over time so they are now statistically rarer than would otherwise be expected. In other words, the error-prone replication process has itself been one of the drivers of evolution to compensate for the deleterious consequences of these errors.
C-to-T transitions in CpG dinucleotides are the most prevalent mutations in human cancers and genetic diseases. These mutations have been attributed to deamination of 5-methylcytosine (5mC), an epigenetic modification found on CpGs. We recently linked CpG>TpG mutations to replication and hypothesized that errors introduced by polymerase ε (Pol ε) may represent an alternative source of mutations. Here we present a new method called polymerase error rate sequencing (PER-seq) to measure the error spectrum of DNA polymerases in isolation. We find that the most common human cancer-associated Pol ε mutant (P286R) produces an excess of CpG>TpG errors, phenocopying the mutation spectrum of tumors carrying this mutation and deficiencies in mismatch repair. Notably, we also discover that wild-type Pol ε has a sevenfold higher error rate when replicating 5mCpG compared to C in other contexts. Together, our results from PER-seq and human cancers demonstrate that replication errors are a major contributor to CpG>TpG mutagenesis in replicating cells, fundamentally changing our understanding of this important disease-causing mutational mechanism.
Main
The emergence and evolution of tumors are driven by mutations, which can be the result of exogenous or endogenous DNA damage or a product of errors during DNA replication1,2. The most common mutation type is a substitution from cytosine to thymine in a CpG dinucleotide (CpG>TpG) across normal somatic and germline cells, as well as cancer cells3,4,5. Germline CpG>TpG mutations are at least ten times more common than expected by chance6 and represent a frequent cause of many genetic diseases7,8. Clustering of cancer mutations into signatures based on the substitution type and context exposed CpG>TpG mutations as the defining feature of somatic single-base substitution signature 1 (SBS1), the most widely observed mutational signature in human cancers and normal cells4. Determining the molecular mechanisms that result in CpG>TpG mutations therefore has important implications for our understanding of evolution in populations as well as in cancer.
The elevated CpG>TpG mutation rate has been linked to 5-methylcytosine (5mC), an epigenetic modification that in humans occurs primarily in CpG dinucleotides9, has an important role in gene regulation and is essential for normal development10. It was observed in vitro that 5mC undergoes spontaneous deamination approximately two times faster than unmodified cytosine11. Moreover, 5mC deamination produces T, resulting in T:G mismatches, which were shown to be repaired much less efficiently than U:G mismatches created by deamination of unmodified cytosines12. CpG>TpG mutations are therefore widely considered to be the result of elevated spontaneous deamination of 5mC.
Surprisingly, we previously observed that CpG>TpG mutations are orders of magnitude more frequent in cancer genomes from individuals with different types of postreplicative mismatch repair (MMR) deficiency or mutations in the exonuclease domain of the major leading-strand DNA polymerase ε (Pol ε), neither of which were thought to be required for the detection or repair of spontaneous deamination13. Instead, MMR and Pol ε ‘proofreading’ through its exonuclease domain are two key components that repair errors introduced during DNA replication14, and their defects cause hypermutated tumors in mice15,16,17,18,19,20, high mutation burden in yeast21,22,23 and the most hypermutated human cancers24,25,26,27,28. This led us to hypothesize that CpG>TpG mutations could also be introduced in a deamination-independent manner as a result of polymerase errors during DNA replication.
Error rates of DNA polymerases have previously been measured using mutation-induced loss of activity of reporter genes (hypoxanthine-guanine phosphoribosyltransferase (HPRT) and lacZ), which can be assayed individually at high scales29,30. However, these methods introduce considerable biases as only certain mutations produce a measurable phenotype, leading to poor representation of sequence contexts. Moreover, the effect of cytosine methylation is difficult to study consistently in such cell-based assays.
Here we set out to directly quantify the misincorporation rate and sequence specificity of mutant and wild-type Pol ε using a sequencing-based approach. To exactly determine which template bases result in what misincorporation, we needed a method that can reliably detect mismatched bases in individual molecules of newly synthesized DNA. Standard genome sequencing cannot be used to detect base changes at single-molecule resolution because they cannot distinguish real variants from technical artifacts introduced during library preparation or from base-calling errors by the sequencing pipeline. Several sequencing-based technologies were recently developed to detect very rare variants, including duplex sequencing31, nanorate sequencing (NanoSeq)32 or bottleneck sequencing system (BotSeqS)33. However, all of them require mutations to be present on both DNA strands, rendering them unsuitable for the direct detection of mismatches introduced by DNA polymerases.
To overcome these limitations, we developed polymerase error rate sequencing (PER-seq), a new method that can detect mismatches introduced by DNA polymerases in a cell-free environment at single-molecule resolution, enabling the quantification of replication errors down to a rate of approximately 1 in 106 replicated bases. We used PER-seq and sequenced over 28 billion bases across more than 130 million molecules to a sufficient depth to detect the misincorporation errors of wild-type and mutant human Pol ε when replicating methylated and unmethylated templates. We show that the sequence-context-specific misincorporation rate of mutant Pol ε measured in vitro closely resembles the mutational signatures observed in tumor samples with combined Pol ε proofreading mutations and MMR deficiency. Strikingly, we detected particularly high Pol ε error rates in a CpG context, which are further increased by the presence of 5mC. Our observations strongly support the hypothesis that CpG>TpG mutations are frequently introduced during DNA replication in a deamination-independent manner.
Tomkova, M., McClellan, M.J., Crevel, G. et al. Human DNA polymerase ε is a source of C>T mutations at CpG dinucleotides. Nat Genet (2024). https://doi.org/10.1038/s41588-024-01945-x
Copyright: © 2024 The authors.
Published by Springer Nature Ltd. Open access.
Reprinted under a Creative Commons Attribution 4.0 International license (CC BY 4.0)
This is an example of the multiple layers of complexity and cumbersome workarounds for 'design' problems that are commonly found in nature because the entire process has been a mindless, near-enough-is-good-enough, make-do-and-mend process; exactly the opposite of what an omniscient, omnipotent, intelligent designer would produce. Quite simply, like an overly complex Heath-Robinson machine, the error-prone complexity of a cell, particularly the genome and its replication, is evidence of unintelligent design.
To ascribe it to a god is to characterize that god as either a bumbling incompetent or a malevolent sadist. It's only by staying willfully ignorant of the details that creationists are being fooled into presenting their god this way, for the commercial benefit and political objectives of their unscrupulous cult leaders.
The Malevolent Designer: Why Nature's God is Not Good
This book presents the reader with multiple examples of why, even if we accept Creationism's putative intelligent designer, any such entity can only be regarded as malevolent, designing ever-more ingenious ways to make life difficult for living things, including humans, for no other reason than the sheer pleasure of doing so. This putative creator has also given other creatures much better things like immune systems, eyesight and ability to regenerate limbs that it could have given to all its creation, including humans, but chose not to. This book will leave creationists with the dilemma of explaining why evolution by natural selection is the only plausible explanation for so many nasty little parasites that doesn't leave their creator looking like an ingenious, sadistic, misanthropic, malevolence finding ever more ways to increase pain and suffering in the world, and not the omnibenevolent, maximally good god that Creationists of all Abrahamic religions believe created everything. As with a previous book by this author, "The Unintelligent Designer: Refuting the Intelligent Design Hoax", this book comprehensively refutes any notion of intelligent design by anything resembling a loving, intelligent and maximally good god. Such evil could not exist in a universe created by such a god. Evil exists, therefore a maximally good, all-knowing, all-loving god does not.
Illustrated by Catherine Webber-Hounslow.
Available in Hardcover, Paperback or ebook for Kindle
Illustrated by Catherine Webber-Hounslow.
The Unintelligent Designer: Refuting The Intelligent Design Hoax
ID is not a problem for science; rather science is a problem for ID. This book shows why. It exposes the fallacy of Intelligent Design by showing that, when examined in detail, biological systems are anything but intelligently designed. They show no signs of a plan and are quite ludicrously complex for whatever can be described as a purpose. The Intelligent Design movement relies on almost total ignorance of biological science and seemingly limitless credulity in its target marks. Its only real appeal appears to be to those who find science too difficult or too much trouble to learn yet want their opinions to be regarded as at least as important as those of scientists and experts in their fields.
Available in Hardcover, Paperback or ebook for Kindle
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