Читать книгу An Introduction to Molecular Biotechnology - Группа авторов - Страница 43

The structure of DNA must be relatively stable and replicated almost flawlessly in order to serve as an information and inheritance carrier. DNA is a relatively stable macromolecule; however, it is liable to constant DNA damage in the body, due to internal or external causes (Table 4.4), which can manifest in mutations. Internal mechanisms are due to spontaneous depurination, deamination, oxidation, and methylation of the DNA bases; external factors include energy‐rich radiation (UV, X‐rays, and radioactivity) and mutagens. Natural mutation rates in bacteria are estimated to be 10^–5 to 10^–6 mutations per gene locus and generation. With eukaryotes these rates are difficult to determine but should also be in the same range.

Table 4.4 Spontaneous DNA damage in a single diploid mammalian cell within 24 hours.

Type of DNA damage	Number in 24 hr
Depurination	18 000
Depyrimidination	600
Cytosine deamination	100
5‐Methylcytosine deamination	10
Oxidation of G to 8‐oxo G	1500
Oxidation of pyrimidines	2000
Methylation of G to 7‐methylguanosine by S‐adenosylmethionine	6000
Methylation of A to 3‐methyladenosine by S‐adenosylmethionine	1200

Source: Alberts et al. (2015). Reproduced with permission of Garland Science.

Mutations where only one or a few nucleotides are exchanged are termed point mutations; other types of mutations include chromosome mutations or rearrangements when larger sequence sections are cut out (deletion) or put in (insertion or translocation), doubled (duplication), or oriented inversely (inversion). If such mutations occur within a transcription unit, they are referred to as gene mutations.

In the human body, nucleotide deamination of nucleotides also spontaneously arises with a rate of 100 deaminations per day and per cell (Figure 4.10; Table 4.4). Cytidine is converted to uracil by deamination. If, following replication, U pairs with A instead of with G, as the original C had done, then the resulting CG pair is completely replaced with a TA pair (Figure 4.11). Further deaminations include: adenosine to hypoxanthine (pairs with C), guanosine to xanthine, and 5‐methylcytosine to thymine (pairs with A). The purine residues guanine and adenine can be removed spontaneously from DNA by hydrolysis (Figure 4.10). Depurination is considered as one of the most common spontaneous mutations and usually leads not only to transversions but also to the deletion of individual bases; over 18 000 purine bases are depurinated daily in every human cell. Under UV radiation (e.g. from extensive sunbathing), neighboring thymine or cytosine residues can be activated, which then form covalently bound dimers (dimerization). The oxidation of guanosine to 8‐oxoguanosine by oxygen radicals (reactive oxygen species[ROS]) can also induce point mutations (Figure 4.10). Therefore, ROS are assumed to play a role in processes, such as aging or cancer.

Figure 4.10 Depurination, deamination, oxidation, and dimerization as examples of major mutation mechanisms.

Figure 4.11 Consequences of deamination, depurination, and oxidation. Cytidine is deaminated, resulting in uracil that then pairs with adenine during replication. If a depurinated nucleotide is not repaired, DNA polymerase passes over the depurinated position during replication. A point deletion that can lead to frameshift mutations results.

In rare cases, the bases can be present in a tautomeric form (Figure 4.12). If they are selected during replication, they can lead to point mutations. G and T are normally present in the keto form and very rarely take the enol form. The amino group of A and C can in rare cases convert into an imino function. Tautomeric adenine pairs with cytosine instead of with thymine; tautomeric thymine pairs with guanine instead of with adenine and vice versa. The resulting nucleotide substitutions fall into the class of the transitions. This includes substitutions of pyrimidine bases with another pyrimidine (T to C or vice versa) and the substitution of a purine base by another (A → G or vice versa). Transversions include the exchange of a purine base with a pyrimidine (A → C or T/G → C or T and vice versa).

Figure 4.12 Base pairing of tautomeric DNA bases. The correct base pairings for A–T and G–C pairs are illustrated in (a). Base pairs between tautomers A, G, C, and T are shown in (b) and (c).

Most of the primary gene changes (deamination, depurination, dimerization, and oxidation) are recognized by repair enzymes (such as AP endonuclease and DNA glycosylases), cut out (as long as the second DNA strand is not also damaged), and repaired by DNA polymerase (especially translesion polymerases) and DNA ligase. In addition, alkyltransferases, photolyases, and incorrect pairing repair and recombination repair systems (see homologous recombination) are also available, which are active after replication. The double helix is also advantageous for any repair process, as genetic information is complementarily saved. Even if the information on one strand is lost, the complementary strand is still available and can be used as a template for the needed correction. In gametes, such as those of humans, thanks to the effectiveness of the repair systems, there are only 10–20 nucleotide substitutions per year in relation to the available 3.2 × 10⁹ bases. The significance of the repair system is easily recognized in humans who are affected by xeroderma pigmentosum, a rare autosomal recessive neurocutaneous disease, in which certain elements of the repair system that are required to repair DNA damage caused by UV radiation do not function. As a result of mutagenic UV radiation from sunlight, aside from numerous neurological and psychiatric symptoms, skin discoloration and skin cancer can occur. This can only be prevented by complete avoidance of sunlight. Table 4.5 lists further diseases caused by defective repair enzymes.

Table 4.5 Genetic diseases, which are associated with defective DNA repair systems.

Syndrome	Phenotype	Damaged proteins
MH2,3,6; MLH1; PMS2	Colon cancer	Mismatch repair
Xeroderma pigmentosum (XP)	Skin cancer, neurological disorders	Excision repair
Cockayne syndrome	UV sensitivity, developmental abnormalities	Coupling of nucleotide excision repair to transcription
XP variant	UV sensitivity, skin cancer	Translesion synthesis by DNA polymerase
BRCA1	Breast and ovarian cancer	Repair through homologous recombination
BRCA2	Breast, ovarian, and prostate cancer	Repair through homologous recombination
Werner syndrome	Premature aging, many tumors	3‐Exonuclease, DNA helicase for DNA repair
Bloom syndrome	Many tumors, stunted growth, genome instability	DNA helicase for DNA repair
Fanconi anemia groups A–G	Malformations, leukemia, genome instability	DNA interstrand cross‐link repair
46 BR‐patient	Hypersensitivity for mutagenic substances, genome instability	DNA ligase I

Source: Alberts et al. (2015). Reproduced with permission of Garland Science.

Owing to the redundant genetic code, not every point mutation in a gene leads to a change in the amino acid sequence. Twenty‐five percent of all theoretically possible substitutions are synonymous, 4% lead to stop codons, and 71% to amino acid exchange. Nucleotide substitutions in the third codon position do not lead to a change in the amino acid in about 69% of cases (referred to as a silent mutation). Should deletions or insertions occur within a coded sequence, a frameshift mutation results, which almost always leads to severe damage of the corresponding proteins (Figure 4.13).

Figure 4.13 Consequences of gene mutations.

Point mutations that cause amino acid exchange (nonsynonymous substitution) often have a negative effect on the corresponding proteins. If the mutation is in the active site or in a binding site, a total loss of function can result. As diploid organisms have at least two copies of every gene, positioned on autologous chromosomes, such a point mutation usually does not lead to physical damage if the other copy of the gene is still intact. Only after both copies have been damaged is there a loss of the corresponding protein function (Figure 4.14). Such disruption is often the basis for disease. This is especially true when the mutation arises in gametes and is then inherited. If the disorder is only in one allele, then it is referred to as a heterozygote character. If both alleles are identical, this is known as a homozygote character (Figure 4.14). In certain genes, such point mutations and the consequences on the health of the individual are widely known. They are referred to as SNPs (single nucleotide polymorphisms), if they occur in several individuals of a population. One of the most important tasks for molecular biotechnologists is the development of diagnostic systems to quickly and reproducibly detect such SNPs. For this purpose mass spectrometry, DNA sequencing, NGS, PCR methods, and DNA chip strategies can be used (see Chapters 13 and 14). This information can help to rationally treat diseases and can lead to a better understanding of their causes.

Figure 4.14 Inheritance of mutations leading to the loss of protein function. Gene A codes for a functional protein; gene a codes for a protein rendered functionless by a mutation. (a) Wild‐type genotype AA. (b) heterozygote genotype Aa. (c) homozygote recessive genotype aa.