An Introduction to Molecular Biotechnology. Группа авторов

Figure 4.8 Schematic summary of DNA replication. SSB, single‐strand binding proteins; Pol III, DNA polymerase III.

Replication begins at specific DNA sequences termed origins. Here the replication bubble opens, and replication occurs in parallel on both the right and left replication forks (Figure 4.9). Whereas in circular bacterial genomes, only one origin of replication is present; in eukaryotic chromosomes a replication start site is positioned on the linear chromosomes every 1000 bp. In this way, even long chromosomes can be replicated in a short time. In eukaryotic cells, four phases are distinguished in a cell cycle: the S phase (DNA synthesis) lasts around eight hours in mammalian cells. The replicated chromatids stay together until the M phase (when mitosis starts). S and M phases are separated by G1 and G2 phases (G for gap). More details of the cell cycle and its measurement can be found in Chapter 18.

Figure 4.9 Asymmetric composition of replication bubbles. DNA is unwound at the origin of replication, and a replication bubble with a right and left replication fork is formed. Replication proceeds in parallel within the replication bubble. DNA primase introduces a complementary RNA primer on each leading strand, so that DNA polymerase III can carry out replication. The individual lagging stands are synthesized as shown in Figure 4.8.

DNA polymerases copy the original nucleotide sequence flawlessly (the error rate during synthesis is one incorrect nucleotide per 10 000 nucleotides). However, special repair enzymes play a large role. Incorrectly paired nucleotides are removed by specific exonucleases and then replaced through DNA polymerase; finally, the phosphoester bond is covalently linked through DNA ligase. Together, these mechanisms reduce mutation rates to less than one in 10 billion nucleotides. Thus, DNA synthesis displays a very high fidelity.

      4.1.5 Mutations and Repair Mechanisms

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.