Underlying genetic variation are changes in DNA sequences. Traditionally, the term mutation has been used in two ways. It can describe an event or process that produces either a change in the base sequence or in the copy number of a DNA sequence, or it can describe the outcome of that process, the altered DNA sequence.
As events, mutations can occur at a wide variety of levels, and can have different con sequences. The altered DNA may contribute to a normal phenotype (such as height) or to a disease phenotype. Very rarely, a mutation has some beneficial effect that produces an altered phenotype conferring a competitive advantage over other phenotypes in the population. The great majority of mutations, however, are neutral: they have no discernible effect on the phenotype. As a result, there has been an increasing trend toward using the more neutral term DNA variant instead of mutation to describe, in a general way, a DNA change produced by mutation. But DNA variants associated with an altered phenotype continue to be widely described as mutations.
DNA variants originate as a result of changes in our DNA that have not been corrected by cellular DNA repair systems. The DNA changes are occasionally induced by radiation and chemicals in our environment, but the great majority arise from endogenous sources: spontaneous errors in normal cellular mechanisms regulating chromosome segregation, recombination, DNA replication, and DNA repair, plus spontaneous chemical damage to DNA.
Mutations are unavoidable. They may have adverse effects on individual organisms, causing aging and underlying many human diseases. But they also provide the raw fuel for natural selection of beneficial adaptations that allow evolutionary innovation and, ultimately, the origin of new species.
Genetic variation arising from endogenous errors in chromosome and DNA function
Natural errors in various processes that affect chromosome and DNA function— chromosome segregation, recombination, and DNA replication—are important contributors to genetic variation. That happens because no cellular function can occur with 100% efficiency—mistakes happen. Endogenous errors in the above processes may often not have harmful consequences, but some of them make important contributions to disease. We will examine in detail how they can cause disease in later chapters; in this section we take a broad look into how they affect genetic variation in general.
DNA replication errors
Each time the DNA of a human diploid cell replicates, six billion nucleotides need to be inserted in the correct order in the newly synthesized DNA molecules. No enzyme is 100% efficient, and DNA polymerases will occasionally make mistakes, inserting the wrong nucleotide to produce mispaired bases (base mismatches). The likelihood of such an error simply reflects the relative binding energies of correctly paired bases and mispaired bases.
In the great majority of cases, the errors are quickly corrected by the DNA polymerase itself. The major DNA polymerases engaged in replicating our DNA have an intrinsic 3′ → 5′ exonuclease activity with a proofreading function. If, by error, the wrong base is inserted, the 3′ → 5′ exonuclease is activated and degrades the newly synthesized DNA strand from its 3′ end, removing the wrongly inserted nucleotide and a short stretch before it. Then the DNA polymerase resumes synthesis again. If mispaired bases are not eliminated by the DNA polymerase, a DNA mismatch repair system is activated (as described below).
Another type of DNA replication error commonly occurs within regions of DNA where there are sequential repeats of a specific nucleotide or short oligonucleotide, such as a run of cytosines or multiple tandem repeats of the CA dinucleotide. When the advancing DNA polymerase encounters a series of short tandem repeats, there will be an increased chance that during DNA replication a mistake is made in aligning the growing DNA strand with its template strand. The two strands can pair-up out of register, causing the growing DNA strand to have fewer or more repeat units (replication slippage—see Figure 1). Errors like this are also often repaired successfully by the DNA mismatch repair system.
Fig1. Insertion and deletion of one or a few nucleotides are often the result of replication slippage at an array of tandem mononucleotide or oligonucleotide repeats. In this example, the parental DNA strand is envisaged to have had 12 tandem CA repeats. After strand separation, a new DNA strand (the nascent or growing strand) is being synthesized, using as a template the DNA strand with the complementary TG repeats. During normal DNA replication, the growing strand often partly dissociates from the template, and then reassociates with the template strand. When there are tandem repeats, mispairing of the growing strand and template strand is facilitated by base pairing between misaligned repeats. (A) The mispairing can cause looping-out of the growing DNA strand, inducing an insertion of, in this case, a single CA dinucleotide, to give a (CA)13 strand. (B) The alternative is that the template strand loops out, inducing a deletion of, in this case, a single CA dinucleotide, to give a (CA)11 strand.
Although the vast majority of DNA changes caused by DNA replication errors are identified and corrected, some persist. That happens because although we have many very effective DNA repair pathways, inevitably DNA repair is also not 100% effective, and unrepaired changes in DNA sequence are an important source of mutations. However, the great majority of the mutations introduced in this way do not cause disease: almost 99% of the genome is noncoding DNA, many mutations in coding DNA do not change an amino acid, and functional noncoding DNA sequences (such as those specifying non coding RNAs) can often tolerate many kinds of sequence change without compromising the function of the sequence.
Chromosome segregation and recombination errors
Errors in chromosome segregation result in abnormal gametes, embryos, and somatic cells that have fewer or more chromosomes than normal, and so have altered numbers of whole DNA molecules. Changes in chromosomal DNA copy number are not uncommon. If they occur in the germline they often cause embryonic lethality or a congenital disorder (such as Down syndrome, which is commonly caused by an extra copy of chromosome 21); changes in copy number of sex chromosomes are more readily tolerated. In somatic cells, changes in chromosomal DNA copy number are a common feature of many cancers.
Various natural errors can also give rise to altered copy number of a specific sequence within a DNA strand that may range up to megabases in length. That can occur by different recombination and recombination-like mechanisms in which non-allelic (but often related) sequences align so that chromatids are paired with their DNA sequences locally out of register, and subsequent crossover or sister chromatid exchange produces chromatids with fewer or more copies of the sequences, for example. The ensuing duplication or deletion of sequences may or may not have functional consequences.
Various endogenous and exogenous sources can cause damage to DNA by altering its chemical structure
DNA is a comparatively stable molecule, but nevertheless there are constant threats to its integrity, causing breakage of covalent bonds within DNA or inappropriate bonding of chemicals to DNA. Most of the damage originates spontaneously within cells (normal cellular metabolism generates some chemicals that are harmful to cells). A minority of the damage is induced by external sources.
Chemical damage to DNA can involve cleavage of covalent bonds in the sugar- phosphate backbone of DNA causing single-strand or double-strand breaks (Figure 2A). Alternatively, bases are deleted (by cleavage of the N-glycosidic bond connecting a base to a sugar; Figure 2B), or they are chemically modified in some way.
Fig2. Four classes of chemical damage to DNA. (A) DNA strand breakage. A single strand may be broken by simple cleavage of a phosphodiester bond (i) or by a more complex single-strand break (ii) where the ends are damaged and sometimes one or more nucleotides are deleted. Double-strand DNA breaks (iii) occur when both strands are broken at sites that are in very close proximity. (B) Base deletion. Hydrolysis cleaves the covalent N-glycosidic bond (shown in deep blue) connecting a base to its sugar. (C) Base modification. Altered bonding or added chemical groups are shown in red. Examples are: 8-oxoguanine (i), which base-pairs to adenine and so induces mutations; thymidine glycol (ii), which is not a mutagen but blocks DNA polymerase; and a DNA adduct (iii) formed by covalent bonding, in this case bonding of an aromatic hydrocarbon, benzo(a)pyrene, to N7 of a guanine residue. (D) Base cross-linking. This involves formation of new covalent bonds linking two bases that may be on the same DNA strand (an intrastrand cross-link) or on complementary DNA strands (an interstrand cross-link). The former includes cyclobutane pyrimidine dimers: linked carbon atoms 4 and 5 on adjacent pyrimidines on a DNA strand (i). This is the most prevalent form of damage incurred by exposure to ultraviolet light from the sun. The anticancer agent cisplatin, (NH3 )2 PtCl2 , causes interstrand cross-links by covalently bonding the N7 nitrogen atoms of guanines on opposite strands (ii).
Many base modifications involve replacing certain groups on bases or adding methyl or larger alkyl groups or other large chemicals (Figure 2C). Sometimes covalent bonds form between two bases (base cross-linking) that may be on the same strand (Figure 2D, part [i]) or on complementary DNA strands (Figure 2D, part [ii]). Chemically modified bases may block DNA or RNA polymerases, causing base mispairing and, if not repaired, mutations.
Endogenous chemical damage to DNA
Most of the chemical damage to our DNA arises spontaneously and is unavoidable. Every day, under normal conditions, around 20,000–100,000 lesions are generated in the DNA of each of our nucleated cells. Three major types of chemical change occur, as listed below. Hydrolytic and oxidative damage are particularly significant, breaking various covalent bonds in the nucleotides of DNA (Figure 3).
• Hydrolytic damage. Hydrolysis is inevitable in the aqueous environment of cells. It can disrupt bonds that hold bases to sugars, cleaving the base from the sugar to produce an abasic site (see Figure 2B); loss of purine bases (depurination) is particularly common. Hydrolysis also strips amino groups from some bases (deamination), leaving a carbonyl (C=O) group. Cytosines are often deaminated to give uracil, which base-pairs with adenine; adenine is occasionally deaminated to produce hypoxanthine, which effectively behaves like guanine by base-pairing with cytosine.
• Oxidative damage. Normal cellular metabolism generates some strongly electrophilic (and, therefore, highly reactive) molecules or ions. The most significant are reactive oxygen species (ROS) formed by the incomplete one-electron reduction of oxygen, including superoxide anions (O2−), hydrogen peroxide (H2 O2 ), and hydroxyl radicals (OH·). ROS are generated in different cellular locations and play important roles in certain intercellular and intracellular signaling pathways, but they mostly originate in mitochondria (where electrons can prematurely reduce oxygen). Endogenous ROS attack covalent bonds in sugars, causing dam age to the sugar–phosphate backbone of DNA. They also attack DNA bases, especially purines (see Figure 11.3), and many derivatives are produced from each base. Some of the base derivatives are highly mutagenic, such as 7,8-dihydro-8- oxoguanine (also called 8-oxoguanine or 8-hydroxyguanine), which base-pairs with adenine; others are not mutagenic but nevertheless block DNA and RNA polymerases (see Figure 2C, parts [i] and [ii]).
• Aberrant DNA methylation. Many cytosines in our DNA are methylated by methyl transferases. Cells also use S-adenosylmethionine (SAM) as a methyl donor in a non enzymatic reaction to methylate different types of molecule, but sometimes SAM can inappropriately methylate DNA to produce harmful bases. Each day about 300–600 adenosines in each nucleated cell are converted to 3-methyladenosine, a cytotoxic base that distorts the double helix, disrupting crucial DNA–protein interactions.
Fig3. DNA sites that are susceptible to spontaneous hydrolytic attack and oxidative damage. Each day, every nucleated human cell loses 5000 or more purines (A and G) and about 300 pyrimidines (C and T) as a result of hydrolytic attack. The N-glycosidic bond connecting a base to its deoxyribose sugar is cleaved by H2O (see Figure 2B for the reaction). In deamination, an amino group is replaced by an oxygen atom to give a carbonyl group. About 100–500 cytosines are replaced by uracil in each cell every day; a smaller number of adenines are replaced by hypoxanthines. In addition, normal metabolism generates reactive oxygen species (see text) that cleave certain chemical bonds not just in bases but also in sugar residues, causing breakage of DNA strands.
Chemical damage to DNA caused by external mutagens
A minority of the chemical damage to our DNA is caused by external agents that can induce mutation (mutagens), including radiation and harmful chemicals in the environment. Ionizing radiation (X-rays, gamma rays, and so on) interacts with cellular molecules to generate ROS that break chemical bonds in the sugar–phosphate backbone, breaking DNA strands (see below). Non-ionizing ultraviolet radiation causes covalent bonding between adjacent pyrimidines on a DNA strand (see Figure 2D, part [i]).
Our bodies are exposed to many harmful environmental chemicals—in our food and drink, and in the air that we breathe. Some chemicals interact with cellular molecules to generate ROS. Other chemicals covalently bond to DNA forming a DNA adduct that may be bulky, causing distortion of the double helix. Cigarette smoke and automobile fumes, for example, have large aromatic hydrocarbons that can bond to DNA (see Figure 2C, part [iii]). Electrophilic alkylating agents can result in base cross-linking.
