Mutagenesis

Mutagenesis (/mjuːtəˈdʒɛnɪsɪs/) is a process by which the genetic information of an organism is changed by the production of 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. A mutagen is a mutation-causing agent, be it chemical or physical, which results in an increased rate of mutations in an organism's genetic code. In nature mutagenesis can lead to cancer and various heritable diseases, and it is also a 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.^[1]

DNA may be modified, either naturally or artificially, by a number of physical, chemical and biological agents, resulting in mutations. Hermann Muller found that "high temperatures" have the ability to mutate genes in the early 1920s,^[2] and in 1927, demonstrated a causal link to mutation upon experimenting with an x-ray machine, noting phylogenetic changes when irradiating fruit flies with relatively high dose of X-rays.^[3][4] Muller observed a number of chromosome rearrangements in his experiments, and suggested mutation as a cause of cancer.^[5][6] The association of exposure to radiation and cancer had been observed as early as 1902, six years after the discovery of X-ray by Wilhelm Röntgen, and the discovery of radioactivity by Henri Becquerel.^[7] Lewis Stadler, Muller's contemporary, also showed the effect of X-rays on mutations in barley in 1928, and of ultraviolet (UV) radiation on maize in 1936.^[8] In 1940s, Charlotte Auerbach and J. M. Robson found that mustard gas can also cause mutations in fruit flies.^[9]

While changes to the chromosome caused by X-ray and mustard gas were readily observable to early researchers, other changes to the DNA induced by other mutagens were not so easily observable; the mechanism by which they occur may be complex, and take longer to unravel. For example, soot was suggested to be a cause of cancer as early as 1775,^[10] and coal tar was demonstrated to cause cancer in 1915.^[11] The chemicals involved in both were later shown to be polycyclic aromatic hydrocarbons (PAH).^[12] 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.^[13] 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, or product molecules resulting from the reaction of DNA and, in this case, cytochrome P450;^[14][15] the mechanism by which the PAH adducts give rise to mutation, however, is still under investigation.

DNA damage is an abnormal alteration in the structure of DNA that cannot, itself, be replicated when DNA replicates. In contrast, a mutation is a change in the nucleic acid sequence that can be replicated; hence, a mutation can be inherited from one generation to the next. Damage can occur from chemical addition (adduct), or structural disruption to a base of DNA (creating an abnormal nucleotide or nucleotide fragment), or a break in one or both DNA strands. Such DNA damage may result in mutation. When DNA containing damage is replicated, an incorrect base may be inserted in the new complementary strand as it is being synthesized (see DNA repair § Translesion synthesis). The incorrect insertion in the new strand will occur opposite the damaged site in the template strand, and this incorrect insertion can become a mutation (i.e. a changed base pair) in the next round of replication. Furthermore, double-strand breaks in DNA may be repaired by an inaccurate repair process, non-homologous end joining, which produces mutations. Mutations can ordinarily be avoided if accurate DNA repair systems recognize DNA damage and repair it prior to completion of the next round of replication. At least 169 enzymes are either directly employed in DNA repair or influence DNA repair processes. Of these, 83 are directly employed in the 5 types of DNA repair processes indicated in the chart shown in the article DNA repair.

Mammalian nuclear DNA may sustain more than 60,000 damage episodes per cell per day, as listed with references in DNA damage (naturally occurring). If left uncorrected, these adducts, after misreplication past the damaged sites, can give rise to mutations. In nature, the mutations that arise may be beneficial or deleterious—this 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, causes the death of the organism. Mutation is also a major source for acquisition of resistance to antibiotics in bacteria, and to antifungal agents in yeasts and molds.^[16][17] In a laboratory setting, 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 functions, 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, but later techniques were developed to introduce specific mutations.

In humans, an average of 60 new mutations are transmitted from parent to offspring. Human males, however, tend to pass on more mutations depending on their age, transmitting an average of two new mutations to their progeny with every additional year of their age.^[18][19]

Mutagenesis may occur endogenously (e.g. spontaneous hydrolysis), through normal cellular processes that can generate reactive oxygen species and DNA adducts, or through error in DNA replication and repair.^[20] Mutagenesis may also occur as a result of the presence of environmental mutagens that induce changes to an organism's DNA. The mechanism by which mutation occurs varies according to the mutagen, or the causative agent, involved. Most mutagens act either directly, or indirectly via mutagenic metabolites, on an organism's DNA, producing lesions. Some mutagens, however, may affect the replication or chromosomal partition mechanism, and other cellular processes.

Mutagenesis may also be self-induced by unicellular organisms when environmental conditions are restrictive to the organism's growth, such as bacteria growing in the presence of antibiotics, yeast growing in the presence of an antifungal agent, or other unicellular organisms growing in an environment lacking in an essential nutrient ^[21][22][23]

Many chemical mutagens require biological activation to become mutagenic. An important group of enzymes involved in the generation of mutagenic metabolites is cytochrome P450.^[24] 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.

While most mutagens produce effects that ultimately result in errors in replication, for example creating adducts that interfere with replication, some mutagens may directly affect the replication process or reduce its fidelity. Base analog such as 5-bromouracil may substitute for thymine in replication. Metals such as cadmium, chromium, and nickel can increase mutagenesis in a number of ways in addition to direct DNA damage, for example reducing the ability to repair errors, as well as producing epigenetic changes.^[25]

Mutations often arise as a result of problems caused by DNA lesions during replication, resulting in errors in replication. In bacteria, extensive damage to 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 damaged sites induces a number of rescue mechanisms that help bypass DNA lesions, however, this may also result in errors. The Y family of DNA polymerases specializes in DNA lesion bypass in a process termed translesion synthesis (TLS) whereby these lesion-bypass polymerases replace the stalled high-fidelity replicative DNA polymerase, transit 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.

Modification of bases

Bases may be modified endogenously by normal cellular molecules. For example, DNA may be methylated by S-adenosylmethionine, thus altering the expression of the marked gene without incurring a mutation to the DNA sequence itself. Histone modification is a related process in which the histone proteins around which DNA coils can be similarly modified via methylation, phosphorylation, or acetylation; these modifications may act to alter gene expression of the local DNA, and may also act to denote locations of damaged DNA in need of repair. DNA may also be 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;^[30] it is, however, uncertain how significant such depurination as caused by the adducts is in generating mutation.

Alkylation and arylation of bases can cause errors in replication. Some alkylating agents such as N-Nitrosamines may require the catalytic reaction of cytochrome-P450 for the formation of a reactive alkyl cation. N⁷ and O⁶ of guanine and the N³ and N⁷ of adenine are most susceptible to attack. N⁷-guanine adducts form the bulk of DNA adducts, but they appear to be non-mutagenic. Alkylation at O⁶ of guanine, however, is harmful because excision repair of O⁶-adduct of guanine may be poor in some tissues such as the brain.^[31] The O⁶ methylation of guanine can result in G to A transition, while O⁴-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.^[32]

Ionizing radiation and reactive oxygen species often oxidize guanine to produce 8-oxoguanine.

See also: Epigenetics

Dimerization

Main article: Dimer

Dimerization consists of the bonding of two monomers to form an oligomer, such as the formation of pyrimidine dimers as a result of exposure to UV radiation, which promotes the formation of a cyclobutyl ring between adjacent thymines in DNA.^[33] 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;^[34] however, individuals with defective DNA repair function, such as those with xeroderma pigmentosum, are sensitive to sunlight and may be prone to skin cancer.

Clinically, whether a tumor has formed as a direct consequence of UV radiation is discernible via DNA sequencing analysis for the characteristic context-specific dimerization pattern that occurs due to excessive exposure to sunlight.^[35]

In the laboratory, mutagenesis is a technique by which DNA mutations are deliberately engineered to produce mutant genes, proteins, or strains of organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the function of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with altered properties, or enhanced or novel functions that may prove to be of use commercially. Mutant strains of organisms that have practical applications, or allow the molecular basis of particular cell function to be investigated, may also be produced.

Early methods of mutagenesis produced entirely random mutations; however, modern methods of mutagenesis are capable of producing site-specific mutations. Modern laboratory techniques used to generate these mutations include:

• Directed mutagenesis
• Site-directed mutagenesis/ PCR mutagenesis
• Insertional mutagenesis
• Signature tagged mutagenesis
• Transposon mutagenesis
• Sequence saturation mutagenesis