Mutagenesis (pron.: /mjuːtəˈdʒɛnɪsɪs/) is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures. In nature mutagenesis can lead to cancer and various heritable diseases, but it is also the driving force of evolution. Mutagenesis as a science was developed based on work done by Hermann Muller, Charlotte Auerbach and J. M. Robson in the first half of the 20th century.
DNA may be modified, either naturally or artificially, by a number of physical, chemical and biological agents, resulting in mutations. In 1927, Hermann Muller first demonstrated mutation with observable changes in the chromosomes can be caused by irradiating fruit flies with X-ray, and lent support to the idea of mutation as the cause of cancer. His contemporary Lewis Stadler also showed the mutational effect of X-ray on barley in 1928, and ultraviolet (UV) radiation on maize in 1936. In 1940s, Charlotte Auerbach and J. M. Robson, found that mustard gas can also cause mutations in fruit flies.
While changes to the chromosome caused by X-ray and mustard gas were readily observable to the early researchers, other changes to the DNA induced by other mutagens were not so easily observable, and the mechanism may be complex and takes longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775, and coal tar was demonstrated to cause cancer in 1915. The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbons (PAH). PAHs by themselves are not carcinogenic, and it was proposed in 1950 that the carcinogenic forms of PAHs are the oxides produced as metabolites from cellular processes. The metabolic process was identified in 1960s as catalysis by cytochrome P450 which produces reactive species that can interact with the DNA to form adducts, the mechanism by which the PAH adducts give rise to mutation however is still under investigation.
DNA may sustain more than 50,000 damages per cell per day, and some estimates put the number of oxidative adducts per cell generated through reactive oxidative species at 150,000. If left uncorrected, these adducts can give rise to mutation. In nature, the mutations that arise may be beneficial or deleterious - it is the driving force of evolution, an organism may acquire new traits through genetic mutation, but mutation may also result in impaired function of the genes, and in severe cases, causing the death of the organism. In the laboratory, however, mutagenesis is a useful technique for generating mutations that allows the functions of genes and gene products to be examined in detail, producing proteins with improved characteristics or novel function, as well as mutant strains with useful properties. Initially the ability of radiation and chemical mutagens to cause mutation was exploited to generate random mutations, later techniques were developed to introduce specific mutations.
Mutagenesis may occur endogenously, for example through spontaneous hydrolysis, or through normal cellular processes that can generate reactive oxygen species and DNA adducts, or through error in replication and repair. Mutagenesis may also arise as a result of the presence of environmental mutagens that induces changes to the DNA. The mechanism by which mutation arises varies according to the causative agent, the mutagen, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on the DNA producing lesions. Some however may affect the replication or chromosomal partition mechanism, and other cellular processes.
Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450. Other enzymes that may also produce mutagenic metabolites include glutathione S-transferase and microsomal epoxide hydrolase. Mutagens that are not mutagenic by themselves but require biological activation are called promutagens.
Many mutations arise as a result of problems caused by the DNA lesions during replication, resulting in errors in replication. In bacteria, extensive damage to the DNA due to mutagens results in single-stranded DNA gaps during replication. This induces the SOS response, an emergency repair process that is also error-prone, thereby generating mutations. In mammalian cells, stalling of replication at a damaged sites induces a number of rescue mechanisms that help bypass DNA lesions, but which also may result in errors. The Y family of DNA polymerases specialize in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymerase, transits the lesion and extend the DNA until the lesion has been passed so that normal replication can resume. These processes may be error-prone or error-free.
DNA is not entirely stable in aqueous solution. Under physiological conditions the glycosidic bond may be hydrolyzed spontaneously and 10,000 purine sites in DNA are estimated to be depurinated each day in a cell. Numerous DNA repair pathway exist for the DNA, however, if the apurinic site failed to be repaired, misincorporation of nucleotide may occur during replication. Adenine is preferentially incorporated by DNA polymerases in an apurinic site.
Cytidine may also become deaminated to uridine at one five-hundredth of the rate of depurination and can result in G to A transition. Eukaryotic cells also contains 5-methylcytosine, thought to be involved in the control of gene transcription, which can become deaminated into thymine.
Bases may be modified endogenously by normal cellular molecules. For example DNA may be methylated by S-adenosylmethionine, and glycosylated by reducing sugars.
Many compounds, such as PAHs, aromatic amines, aflatoxin and pyrrolizidine alkaloids, may form reactive oxygen species catalyzed by cytochrome P450. These metabolites form adducts with the DNA, which can cause errors in replication, and the bulky aromatic adducts may form stable intercalation between bases and block replication. The adducts may also induce conformational changes in the DNA. Some adducts may also result in the depurination of the DNA, it is however uncertain how significant such depurination as caused by the adducts is in generating mutation.
Some alkylating agents such as N-Nitrosamines may also require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. Alkylation and arylation of bases can cause errors in replication. N and O of guanine and the N and N of adenine are most susceptible to attack; while N-guanine adducts, which form the bulk of DNA adducts, appear to be non-mutagenic, alkylation at O of guanine is harmful because excision repair of O-adduct of guanine may be poor in some tissues such as the brain. The O methylation of guanine can result in G to A transition, while O-methylthymine can be mispaired with guanine. The type of the mutation generated however may be dependent on the size and type of the adduct as well as the DNA sequence.
Ionizing radiations and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.
Some alkylating agents may produce crosslinking of DNA. Some natural occurring chemicals may also promotes crosslinking, such as psoralens after activation by UV radiation, and nitrous acid. Interstrand cross-linking is more damaging as it blocks replication and transcription and can cause chromosomal breakages and rearrangements. Some crosslinkers such as cyclophosphamide, mitomycin C and cisplatin are used as anticancer chemotherapeutic because their high degree of toxicity to proliferating cells.
UV radiation promotes the formation of a cyclobutyl ring between adjacent thymines, resulting in the formation of pyrimidine dimers. In human skin cells, thousands of dimers may be formed in a day due to normal exposure to sunlight. DNA polymerase η may help bypass these lesions in an error-free manner; however, individuals with defective DNA repair function, such as sufferers of Xeroderma pigmentosum, are sensitive to sunlight and may be prone to skin cancer.
The planar structure of chemicals such as ethidium bromide and proflavine allows them to insert between bases in DNA. This insert causes the DNA's backbone to stretch and makes slippage in DNA during replication more likely to occur since the bonding between the strands is made less stable by the stretching. Forward slippage will result in deletion mutation, while reverse slippage will result in an insertion mutation. Also, the intercalation into DNA of anthracyclines such as daunorubicin and doxorubicin interferes with the functioning of the enzyme topoisomerase II, by tightening the DNA's strands by the stretching, making replication as well as causing mitotic homologous recombination.
Ionizing radiations may produce highly reactive free radicals that can break the bonds in the DNA. Double-stranded breakages are especially damaging and hard to repair, producing translocation and deletion of part of a chromosomes. Alkylating agents like mustard gas may also cause breakages in the DNA backbone. Oxidative stress may also generate highly reactive oxygen species that can damage the DNA. Incorrect repair of other damages induced by the highly reactive species can also lead to mutations.
Transposon and virus may insert DNA sequence into coding region or functional elements of a gene and result in inactivation of the gene.
While most mutagens produce effects that ultimately result in error in replication, some mutagens may affect directly the replication process. Base analog such as 5-bromouracil may substitute for thymine in replication. Some metals such as cadmium, chromium, and nickel may alter the fidelity of DNA replication.
Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organism. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutants strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.
Early approaches to mutagenesis rely on methods which are entirely random in the mutations produced. Cells may be exposed to UV radiation or mutagenic chemicals, and mutants with desired characteristic are then selected. For example, Escherichia coli may be exposed to UV radition, then plated onto agar medium. The colonies formed are then replica-plated, one in rich medium, another in minimal medium, and mutants that have specific nutritional requirement can then be identified by its inability to grow in minimal medium and isolated.
A number of methods for generating random mutation in specific protein were later developed to screen for mutants with interesting or improved properties. This may be done by using doped nucleotides in oligonucleotides synthesis, or conducting a PCR reaction in conditions that enhance misincorporation of nucleotide, thereby generating mutants.
It is desirable that specific changes can be introduced to the DNA. Analogs of nucleotides and other chemicals were first used to generate localized point mutations. Such chemicals may be aminopurine which induces AT to GC transition, while nitrosoguanidine, bisulfite, and N-hydroxycytidine may induce GC to AT transition. These technique allows specific mutations to be engineered into a protein, however, they are not flexible in the kinds of mutants generated.
Current techniques for site-specific mutation commonly involve using mutagenic oligonucleotides in a primer extension reaction with DNA polymerase. This methods allows for point mutation, or deletion or insertion of small stretches of DNA to be introduced at specific sites. Advances in methodology have made such mutagenesis now a relatively simple and efficient process.
The site-directed approach may be done systematically in such technique as alanine scanning mutagenesis whereby residues are systematically mutated to alanine in order to identify residues important to the structure or function of a protein.
Combinatorial mutagenesis is a technique whereby large number of mutants may be screened for a particular characteristic. In this technique, a few selected positions or a short stretch of DNA may be exhaustively modified to obtain a comprehensive library of mutant proteins. One approach of this technique is to excise a portion of DNA and replaced with a library of sequences containing all possible combinations at the desired mutation sites. The segment may be at an enzyme active site, or sequences that have structural significance or immunogenic property. A segment however may also be inserted randomly into the gene in order to assess the structural or functional significance of particular part of protein.
In cancer research engineered mutations also provide mechanistic insights into the development of the disease. Insertional mutagenesis using transposons, retrovirus such as mouse mammary tumor virus and murine leukemia virus may be used to identify genes involved in carcinogenesis and to understand the biological pathways of specific cancer. Various insertional mutagenesis techniques may also be used to study the function of particular gene.
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