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Mutation is a heritable or somatic change in the nucleotide sequence of the genome of an organism, a virus, or extrachromosomal DNA or RNA. Mutations arise from errors in DNA replication, from spontaneous chemical changes in DNA, from damage by physical and chemical agents, and from the activity of mobile genetic elements. They are the ultimate source of new alleles, and therefore of the heritable variation on which natural selection and genetic drift act in population genetics and evolutionary biology. A mutation is not an "error" in any evaluative sense. Most mutations that affect fitness are harmful, some are neutral, and a few are beneficial, and which category applies depends on the environment and the genetic background.

Mutation is random with respect to fitness, in the sense that the probability that a mutation occurs does not depend on whether it would be useful to the organism. This was shown by the Luria-Delbrück experiment (1943) and by replica plating (Lederberg and Lederberg, 1952). Mutation is not random in every respect. Rates differ among sites, types of change, genes, and species, and the chemistry of the bases makes some changes much more likely than others.

Spontaneous mutations arise in the absence of external mutagens. They result from the limits of the accuracy of enzymes and from the intrinsic chemistry of nucleic acids in water.

Replication errors and tautomers

A DNA polymerase selects the correct nucleotide by the geometry of the base pair in its active site, and it inserts a wrong nucleotide at a rate of about 10⁻⁴ to 10⁻⁵ per base. Bases can exist in rare tautomeric forms, for example the enol form of guanine or thymine and the imino form of cytosine or adenine. A base in a rare tautomeric form can pair with the wrong partner. Watson and Crick proposed in 1953 that this could cause mutations. For example, a cytosine in the imino form can pair with adenine. Structural studies have shown that mispairs can also form by shifts to the wobble geometry and by ionized bases, so tautomerism is one of several routes, and its relative contribution is still under study. The tautomerization is a fast proton shift and not a quantum "restructuring" in any special sense.

Errors are corrected in two steps. Many replicative polymerases have a 3′ to 5′ exonuclease domain that removes a mismatched nucleotide at the primer end (proofreading), which improves accuracy by about 100-fold. After replication, the mismatch repair system recognizes mismatches and removes the newly made strand, which gives another improvement of about 100-fold. The errors that remain become permanent mutations after the next round of replication. The combined effect gives a final error rate of about 10⁻⁹ to 10⁻¹⁰ per base pair per replication in many cells:

Replication slippage in repeated sequences produces insertions and deletions.

Chemical damage

• Deamination. Cytosine can lose its amino group to become uracil, which pairs with adenine, and a C:G pair becomes a T:A pair after replication unless uracil is removed by uracil-DNA glycosylase. Deamination of 5-methylcytosine gives thymine directly, which is not recognized as foreign, so CpG dinucleotides are mutation hotspots in vertebrates, with C to T transitions at about ten times the rate at other sites.
• Depurination. The bond between a purine and the sugar can hydrolyze spontaneously, which leaves an abasic site. A mammalian cell loses several thousand purines per day.
• Oxidative damage. Reactive oxygen species from metabolism oxidize guanine to 8-oxoguanine, which can pair with adenine and cause G to T transversions.

Physical and Chemical Mutagens

Mutagens increase the mutation rate:

• Ultraviolet radiation is non-ionizing, but UV-B and UV-C are absorbed by DNA and cause covalent bonds between adjacent pyrimidines on the same strand. This forms cyclobutane pyrimidine dimers and (6-4) photoproducts, which distort the helix and block replicative polymerases. Bypass by error-prone translesion synthesis polymerases, such as polymerase eta, produces characteristic C to T and CC to TT changes, a signature found in skin cancers. Defects in nucleotide excision repair cause xeroderma pigmentosum.
• Ionizing radiation (X-rays and gamma rays) damages DNA directly and by producing hydroxyl radicals from water. It causes base damage, single-strand breaks, and double-strand breaks, which lead to deletions and rearrangements. Hermann Joseph Muller showed in 1927 that X-rays cause mutations in Drosophila. Repair of double-strand breaks by non-homologous end joining is error-prone in many cases, with small insertions or deletions at the junction, but it often rejoins ends without change, so it is not mutagenic in every case. Homologous recombination is the more accurate route.
• Intercalating agents, such as ethidium bromide and acridine dyes (proflavin), are flat aromatic molecules that insert between stacked base pairs. They lengthen and unwind the helix, and they cause insertions or deletions of one base pair (frameshifts), shown by the work of Brenner, Crick, and Streisinger on bacteriophage T4.
• Other chemicals. Base analogs (5-bromouracil) are incorporated and mispair, alkylating agents (ethyl methanesulfonate) change base pairing, and polycyclic aromatic hydrocarbons and aflatoxin B₁ form bulky adducts after metabolic activation. The Ames test measures mutagenicity by the reversion of histidine-requiring Salmonella.

Point mutations

A point mutation changes one nucleotide. A change between two purines (A and G) or two pyrimidines (C and T) is a transition, and a change between a purine and a pyrimidine is a transversion. There are twice as many possible transversions as transitions, but transitions are more frequent, and the observed transition to transversion ratio is often about 2 for nuclear DNA. Reasons include the frequent deamination of cytosine and 5-methylcytosine and the fact that transition mispairs (such as G:T) fit the polymerase active site better than transversion mispairs. Transversions are not "guarded against" by special mechanisms. They are made less often.

Effects in coding sequences:

• A synonymous mutation does not change the amino acid, because the code is degenerate. It is not always neutral. It can alter the speed of translation, RNA splicing, mRNA stability, and folding, and codon usage matched to tRNA abundance affects expression.
• A missense mutation changes one amino acid. The effect depends on the position and the chemical similarity of the new residue. Replacing leucine by valine is often tolerated. In sickle cell disease, the replacement of glutamic acid by valine at position 6 of the β-globin chain creates a hydrophobic patch on the surface of hemoglobin S, which makes deoxygenated molecules polymerize into fibers. The mechanism is aggregation (a change in quaternary interactions), and not a general misfolding of the tertiary structure.
• A nonsense mutation creates a premature stop codon. It usually truncates the protein, and the mRNA is often degraded by nonsense-mediated decay.
• Mutations can also change start codons, splice sites, and regulatory sequences.

Insertions and deletions

An indel is the insertion or deletion of one or more nucleotides. In a coding region, an indel whose length is not a multiple of three causes a frameshift mutation, which changes the reading frame downstream and usually creates a premature stop codon. An indel that is a multiple of three keeps the frame. Expansions of trinucleotide repeats cause diseases such as Huntington's disease (CAG) and fragile X syndrome (CGG).

Structural and chromosomal mutations

Larger changes include deletions, duplications, inversions, translocations, and changes in chromosome number (aneuploidy and polyploidy). They arise from errors in repair of double-strand breaks, from non-allelic homologous recombination between repeats, from transposable elements, and from errors in meiosis and mitosis. A translocation can create a fusion gene, as in the BCR-ABL1 fusion of the Philadelphia chromosome in chronic myeloid leukemia. Gene duplication is a main source of new genes, because one copy can keep the original function while the other changes (neofunctionalization).

Germline and somatic mutations

Germline mutations occur in cells that give rise to gametes and can be inherited. Somatic mutations occur in other cells, are not passed to offspring, and accumulate during life, causing cancer and mosaicism.

Loss-of-Function, Gain-of-Function, and Epistasis

A mutation affects the phenotype only if it alters the amount, timing, or activity of a gene product. Coding changes are described above. Mutations in enhancers, promoters, and other regulatory sequences change the binding of transcription factors and so the pattern and level of expression, without changing the protein. Such changes are important in evolution, for example in changes of the regulation of developmental genes. Mutations are also described as loss-of-function, gain-of-function, or dominant-negative, according to their effect on the gene product, and their effects interact with each other (epistasis).

Mutation rates

Rates are about 10⁻⁶ to 10⁻⁴ per base per replication cycle in RNA viruses, about 2 × 10⁻¹⁰ per base per generation in Escherichia coli, and about 1 to 1.5 × 10⁻⁸ in humans. A human child has about 60 to 70 new single-nucleotide variants, and the number increases with the age of the father. For a genome of L sites and a rate μ per site, the number of new mutations is approximately Poisson distributed with mean λ = L μ. For L = 6 × 10⁹ (the diploid human genome) and μ = 1.2 × 10⁻⁸, λ ≈ 72. The population mutation parameter for a diploid population is θ = 4_N__eμ, where N_e is the effective population size.

Fate

The distribution of fitness effects of new mutations differs among species, but most non-synonymous mutations are harmful, a substantial fraction are neutral or nearly neutral, and few are beneficial. A new neutral mutation in a diploid population is fixed with probability 1/(2_N_), and a new beneficial mutation with a small advantage s with probability of about 2_s_, so that most beneficial mutations are lost by drift while rare. This is the basis of the neutral theory of molecular evolution.

Excision, Mismatch, and Recombinational Repair

Cells reduce the mutation rate by DNA repair: base excision repair, nucleotide excision repair, mismatch repair, direct reversal, homologous recombination, and non-homologous end joining. Defects cause high mutation rates and disease, such as Lynch syndrome (mismatch repair).

How did the Luria-Delbrück fluctuation test prove that mutations occur randomly prior to selection?

In 1943, Salvador Luria and Max Delbrück grew parallel independent cultures of *Escherichia coli* in phage-free broth before plating them onto agar coated with lethal bacteriophage T1. If phage resistance were physiologically induced by exposure to the virus (Lamarckian post-adaptation), the number of resistant colonies per plate would follow a Poisson distribution where variance equals the mean. Instead, they observed "jackpot" fluctuations (variance >> mean), proving that resistance mutations arose spontaneously at random generations prior to viral exposure.

Why are CpG dinucleotides major mutation hotspots for C-to-T transitions in vertebrate genomes?

In vertebrate DNA, cytosine bases followed by guanine (5'-CpG-3') are frequently methylated at the 5-carbon position by DNA methyltransferases to regulate gene expression. Whereas spontaneous hydrolytic deamination of unmethylated cytosine yields uracil—an unnatural DNA base immediately recognized and excised by uracil-DNA glycosylase—deamination of 5-methylcytosine yields canonical thymine (5-methyluracil). Because DNA repair enzymes cannot easily distinguish this newly formed thymine from normal template thymines, the T:G mismatch frequently escapes repair and fixes as a C:G → T:A transition.

Why does paternal age strongly increase the number of de novo germline mutations in human offspring?

In human females, oogonia complete all mitotic divisions before birth and arrest in meiosis I, undergoing roughly 22 to 24 cell divisions regardless of maternal age at conception. Conversely, male spermatogonial stem cells divide continuously via mitosis every 16 days (~23 divisions per year) after puberty. A 20-year-old father's sperm has undergone ~150 mitotic divisions, whereas a 40-year-old father's sperm has undergone ~610 divisions, adding approximately 1.5 to 2 new single-nucleotide mutations per year of paternal age.

• Genetic variation
• DNA repair
• Mutagen
• Neutral theory of molecular evolution
• Population genetics
• Mutation rate