Читать книгу An Introduction to Molecular Biotechnology - Группа авторов - Страница 42

With every cell division (mitosis; Figure 4.7), the entire genome of the cell is duplicated. This means that two identical chromatids result from every chromosome. These chromatids are identical daughter chromosomes that will be distributed to the daughter cells following cell division. The duplication of the DNA, which is referred to as DNA replication, occurs in a semiconservative manner.

During semiconservative replication, the DNA double strand is locally separated into single strands, and a replication fork is formed. The single strands serve as a matrix for the synthesis of each complementary new strand. The DNA replication is a complex process, in which many proteins and enzymes are involved (Figure 4.8). To open the double strands, a helicase is needed. The leading strand, orientated in the 3′ → 5′ direction, can be copied directly by the DNA polymerase, as synthesis occurs in a 5′ → 3′ direction. The opposing strand, termed lagging strand, cannot be copied in the same way as it is orientated in a 5′ → 3′ direction. As soon as the DNA is present as single strands, specific proteins bind (single‐strand binding proteins) and prevent the reformation of the double helix. DNA primase places short RNA primers, which are complementary to the DNA sequence, at regular intervals on the lagging strand. These RNA primers can be lengthened by DNA polymerase until the next RNA primer is reached (referred to as Okazaki fragments). The RNA is then removed and replaced by deoxynucleoside triphosphates (dNTPs). The Okazaki fragments are linked through the DNA ligase. A sliding clamp keeps the DNA polymerase firmly on the DNA when it is moving. An ATP‐depending clamp loader protein complex supports this process. Enzymes involved in replication vary between prokaryotes and eukaryotes. However, the general composition of the multienzyme complex is similar. During the unwinding of the double helix, DNA topoisomerases cut the DNA at regular intervals to prevent the rotation of the double helix. DNA topoisomerase I catalyzes single‐strand breaking, while DNA topoisomerase II can cut both DNA strands at the same time.

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.