Mutagenesis is the process by which an organism's deoxyribonucleic acids (DNA) change, resulting in a gene mutation. A mutation is a permanent and heritable change in genetic material, which can result in altered protein function and phenotypic changes. DNA consists of nucleotides that contain a phosphate backbone, a deoxyribose sugar, and one of four nitrogen-containing bases (adenine [A], guanine [G], cytosine [C], and thymine [T]). DNA mutagenesis occurs spontaneously in nature or as a result of mutagens (agents with a predisposition to alter DNA). Furthermore, molecular genetic techniques, such as polymerase chain reaction (PCR), have revolutionized how mutations are obtained and studied. Mutagenesis is the driving force of evolution; however, it can also lead to cancers and heritable diseases.
Single base-pair substitutions are the most common cause of human pathology. An example is sickle cell disease, where a single base-pair mutation results in glutamate to valine amino acid substitution. Otherwise, human disease is a result of a mixed assortment of insertions, deletions, duplications, inversions, expansions, fusions, and complex rearrangements. For example, a CGG repeat expansion in the FRM1 gene causes fragile X syndrome, while a fusion protein BCR-ABL results in chronic myeloid leukemia (CML).
It is noteworthy that not all DNA mutations will have an impact on protein synthesis or function. There are various types of mutations, such as silent, missense, nonsense, and frameshift mutations.
A silent mutation is a nucleotide substitution that codes for the same amino acid; therefore, there is no change in the amino acid sequence or protein function. A missense mutation is when a nucleotide substitution results in an amino acid change.
Missense mutations have variable effects but can lead to a decreased or altered protein function (e.g., sickle cell disease).
A nonsense mutation is when a nucleotide substitution results in a new stop codon, which includes UGA, UAA, and UAG (remember the mnemonic "U Go Away, U Are Away, and U Are Gone," respectively). These protein products are truncated and frequently nonfunctional (e.g., cystic fibrosis).
A frameshift mutation occurs through the addition or deletion of nucleotides not divisible by 3, resulting in the misreading of the downstream nucleotides. These proteins may be shorter or longer, and protein function may disrupt or altered (e.g., Duchenne muscular dystrophy).
Other types of mutations exist outside of the coding sequence. These include splice site, promoter or enhancer sequence, and termination site mutations. For example, in some forms of B-thalassemia, a mutated splice site results in the use of cryptic splice locations, causing impaired B-globin synthesis.
Mutagenesis is the driving force behind evolution, and genetic variation is the result of mutations. Any change in genetic information may result in advantageous or disadvantageous phenotypic characteristics that impact an organism's fitness. When a mutation results in a higher fitness, natural selection favors these phenotypes, and these traits are more likely to be passed to offspring.
Mutagenesis occurs as a result of DNA replication errors, DNA damage, and lab techniques. Here we will break mutagenesis down into endogenous and exogenous causes.
Mutagenesis is a technique used in molecular biology to create mutant genes, proteins, and organisms. Two primary mutagenesis techniques are random-and-extensive mutagenesis (REM) and site-directed mutagenesis (SDM). The methods accomplished by polymerase chain reaction (PCR) and non-polymerase chain reaction (non-PCR). Other methods of mutagenesis, including CRISPR/Cas9 technology, TALENs (transcription activator-like effector nucleases), and Zinc-Finger Nucleases (ZFN).
Site-directed mutagenesis. SDM is a technique where DNA can be modified at a specific nucleotide location, causing a predetermined amino acid change. These substitution mutations can result in drastic changes in protein conformation and function. This technique requires (1) a DNA template with a target gene, (2) knowledge of the target gene's nucleotide sequence, and (3) a short primer (commonly 20 to 30 base pairs) complementary to the target sequence that is modified to contain a mismatched nucleotide (typically 1 to 3 base pairs that will cause an amino acid change). The general procedure for SDM is the following:
Techniques for performing SDM:
PCR and non-PCR techniques are in vitro methods for performing SDM. In vitro synthesis has four essential components: DNA template, modified primers, deoxyribonucleic nucleoside triphosphates (dNTPs [i.e., dATP, dCTP, dGTP, dTTP]), and DNA polymerase (thermostable or thermolabile). The polymerase chain reaction technique utilizes thermostable DNA polymerases (e.g., Taq, Pfu, and Vent), at least two different primers, and multiple heating and cooling cycles (average 30 cycles). Each cycle has three phases: denaturation (approximately 95 degrees Celsius), annealing (approximately 55 degrees Celsius), and extension (approximately 72 degrees Celsius). The denaturation phase is when the template DNA molecule separates into two single-stranded molecules. The annealing phase is when the modified primer base pairs at the sequence of interest. Lastly, the extension phase is when the annealed primers extended according to the template strand. The advantage of PCR is that it works more favorably with different DNA templates (i.e., single or double-stranded DNA and GC-rich regions) and produces millions of copies of a target gene. The disadvantage is there is less sequence fidelity (i.e., thermostable polymerases are more error-prone in comparison to thermolabile polymerases). The non-polymerase chain reaction technique utilizes thermolabile DNA polymerases, one primer, and constant reaction temperature (e.g., 37 degrees Celsius). In this strategy, the DNA template denatured with an alkali solution or heat. The template annealed with the modified primer at room temperature. Though single or double-stranded DNA templates can use, more favorable outcomes occur if a single-stranded DNA template is used (this increases the success of annealing). DNA synthesis carried out at 37 degrees Celsius. It produces a hybrid DNA molecule (i.e., one template strand and one newly synthesized mutant strand), which then transformed or infected into E. coli. Once in E.coli, the mutant DNA and wild-type DNA can be segregated. Non-PCR has greater sequence fidelity than the PCR method; however, there is only one DNA strand generated (rather than millions).
Screening for desired mutants in SDM:
There are different methods for selecting and screening for desired mutants. One of PCR approach is to use plasmids that are grown in E. coli (these are dam methylated) and contain a gene for antibiotic resistance. As discussed, modified forward and reverse primers are extended around plasmid DNA, creating copies of the template with the inserted mutations. Since the original parental template strand is methylated, a restriction endonuclease Dpn I - specific for methylated and hemimethylated DNA - digests this DNA leaving only the mutant plasmid, which further amplified. The plasmid transformed into E.coli, and bacteria are grown on media that contain the appropriate antibiotic for the plasmid. One can then pick single colonies and grow them overnight to isolate the mutant plasmid DNA. Please refer to the article by Bachman for a more detailed review. Since there is the possibility of random mutations in PCR and non-PCR methods, sequencing performed to verify the desired mutation made. DNA sequencing technology (e.g., Sanger sequencing or next-generation sequencing) can utilize to determine nucleotide sequences.
Random and extensive mutagenesis. REM is a useful approach when many mutations desired; however, there is less control over the resulting modifications. This technique has helped map out critical residues of proteins (e.g., EcoRV restriction endonuclease). REM can accomplish through different methods:
Insertions and deletions. PCR is effective at producing insertions or deletions. Small insertions or deletions (less than ten base pairs) can occur by utilizing the modified primer design described above. Large deletions can occur by joining two PCR-amplified fragments, which omits a portion of a DNA fragment. Large insertions can occur using megaprimers or overlap-extension PCR.
CRISPR-Cas9. This technology is a genome-editing tool derived from bacterial defenses against viruses and foreign plasmids. It has two vital components: a guide RNA (gRNA) that binds complementary to a target DNA sequence, and an endonuclease (Cas9) that causes a dsDNA break that requires repair. Cas9-induced site-specific dsDNA breaks induce endogenous cell repair mechanisms, which can exploit to modify DNA. The error-prone nonhomologous end-joining (NHEJ) can rapidly ligate DNA breaks; however, it may generate small insertions and deletions that can cause frameshift mutations. Alternatively, dsDNA breaks can be repaired by homology-directed repair (HDR), which can insert exogenous DNA and introduce precise genome editing.
TALENS vs. Zinc finger nucleases. TALENs and ZFN are gene-editing tools with similar principles; however, different underlying designs. They both are proteins that consist of DNA binding domains (i.e., TALE proteins vs. zinc fingers domains) and DNA cleavage domains (e.g., Fok1). DNA specificity resides in the TALE proteins and the zinc finger domains. For cleavage to occur, there must be dimerization of the restriction enzyme; therefore, at least two proteins needed per target DNA. Once dimerization occurs at a target location, a double-stranded DNA break made. Similar to CRISPR, this repaired by NHEJ (with the possibility of insertions and deletions) or homologous recombination (where exogenous DNA can add). A key difference between TALENs and ZFN is that TALE proteins recognize a single base, while zinc finger domains recognize 3 or 4 bases. Given zinc fingers must recognize multiple bases, it may reduce DNA specificity in comparison to TALE proteins.
Mutagenesis has multiple implications in clinical medicine. This article will discuss a few, including carcinogenesis, heritable diseases, microorganism resistance, large scale mutagenic projects, and precision medicine.
Carcinogenesis (i.e., tumorigenesis or oncogenesis) is when a cell begins to divide uncontrollably. Mutations of oncogenes (which promote cell growth), tumor suppressor genes (which inhibit cell growth), or cell-cycle genes (which regulate the cell-cycle) can generate a clonal cell population with highly proliferative properties, leading to cancer. Approximately two-thirds of cancer driver mutations are attributable to spontaneous mutagenesis that occurs during normal DNA replication. Furthermore, multiple agents (such as those described in the "mechanism" section) are known to be associated with carcinogenesis. Tobacco contains DNA methylating agents, alkylating agents, polycyclic aromatic hydrocarbons, and N-nitrosamines, among others. Smoking tobacco is proven to increase the risk of a multitude of cancers, including lung, colorectal, head and neck, and lower urinary tract cancers. Tobacco accounts for 16% of all cancer diagnoses and approximately one-third of all cancer deaths in the US. It highlights why counseling on smoking cessation, and other preventative health measures, becomes so important.
Ionizing radiation (IR), as a consequence of atomic radiation (e.g., Chernobyl) or iatrogenic causes, is associated with papillary thyroid carcinoma (PTC). In PTC, IR increases the rate of DNA rearrangements that cause a permanently active promotor region of the tyrosine kinase, ret (leading to PTC pathogenesis). Accordingly, anti-promoter agents may one day be a method for future therapy.
Interestingly, chemotherapeutic drugs used to combat cancers - in particular, cisplatin, cyclophosphamide, and etoposide - are themselves carcinogenic. These drugs have implicated in the development of tumor resistance and subsequent cancer development. Therefore, care should be taken when choosing treatment regimens, especially in childhood cancers.
Heritable diseases have their origin in mutagenesis, as these are permanent DNA changes that passed to offspring. Some examples of hereditary diseases include sickle cell disease, Tay-Sachs disease, cystic fibrosis, Huntington disease, Duchenne muscular dystrophy, and hemophilia, among many others. A well-studied example is sickle cell anemia. It is an autosomal recessive disease resulting from a missense mutation in the B-globin gene (i.e., a point mutation resulting in a glutamic acid to valine substitution). Research shows that this mutation created an adaptive advantage in heterozygote carriers in malaria-endemic areas. However, homozygous inheritance has a poor prognosis due to increased risk for anemia, infection, stroke, and organ damage.
Drug resistance in microorganisms is a result of mutagenesis. Multidrug-resistant (MDR) organisms are associated with increased antibiotic use. MDR organisms have increased mortality rates, and it estimated that by 2050 there might be 300 million premature deaths worldwide due to MDR organisms. It highlights the need to practice effective antimicrobial stewardship to slow the development of MDR organisms.
An exciting example of drug resistance can occur in the treatment of Chagas disease. Benznidazole is a front-line pro-drug used in Chagas treatment. New evidence suggests that Trypanosoma cruzi may process this drug into metabolites that cause whole-genome mutations. Therefore, this may be a reason for treatment failure and the development of drug-resistant parasites. It can even lead to resistance to different classes of drugs, like posaconazole, used in combination therapy of Trypanosoma cruzi. Therefore, vigilance is required when using benznidazole in combination therapy.
Large scale mutagenesis techniques can use to map functionally important amino acid residues that affect particular phenotypes. For example, a mouse study on the Golgi protein GMAP-210 showed that gene alteration caused a phenotype that resembled human achondrogenesis type 1A. It helped lead to the discovery of a similar mutation found in humans.
Lastly, precision medicine (PM) is a concept where disease treatment based on knowing an individual's genome abnormalities, which became relevant as whole-genome sequencing more accessible. PM is dependent on new therapeutic strategies, drug development, and gene-oriented treatment. For example, gene-targeted techniques like CRISPR/Cas9 and TALENs may one day have clinical use. CRISPR investigated to treat single-gene mutations (e.g., DMD, cystic fibrosis), HIV, and cancers. There are still multiple challenges to PM, such as validating functional mechanisms of mutated gene expression. However, PM may one day have an essential impact on disease cures.
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