Fundamentals of Molecular Biology. Part 2: Molecular Genetic Mechanisms - A. N. Ogurtsov 2011

DNA Replication and Repair
Replication Models

Following Watson and Crick's elucidation of the DNA double helix Structure, it was hypothesized that the synthesis of new helices occurs by using existing (parental) strands as a template to form daughter strands complementary to the parental ones.

Theoretically, such a model of template-directed nucleotide pairing can be achieved via either a conservative or a semi-conservative mechanism.

According to the conservative mechanism, two daughter strands could form a new DNA double helix, or duplex, while the parental duplex remains entirely intact.

According to the semi-conservative mechanism, the strands of the parental DNA double helix completely unwind and separate, with each strand serving as a template to synthesize a complementary daughter strand through base pairing.

Evidence that DNA replicates via a semi-conservative mechanism was obtained in a landmark experiment conducted by Matthew Meselson and Franklin Stahl in 1958, illustrated in Figure 50.

Initial E. coli Cells were grown in a medium containing only the "heavy" nitrogen isotope ¹⁵N, so that all original DNA molecules were "labeled" with this isotope.

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Figure 50 - Schemes of conservative and semi-conservative mechanisms of METABOLISM/36.html">DNA Replication

Subsequently, the cells were transferred to a medium containing only the "light" nitrogen isotope ¹⁴N. Samples were periodically harvested, and the DNA from each sample was analyzed by equilibrium density gradient centrifugation. This made it possible to detect heavy-heavy (H-H), heavy-light (H-L), and light-light (L-L) duplexes.

If replication followed the conservative mechanism, The formation of H-L duplexes would be impossible, whereas the semi-conservative mechanism predominantly generates H-L duplexes (Figure 50). The experiment demonstrated that the first filial generation consists entirely of H-L duplexes, while the second generation (specifically, the 1.9th generation) contains approximately equal proportions of H-L and L-L duplexes (Figure 51). In subsequent generations, an increasing proportion of L-L duplexes appeared, exactly as predicted by the semi-conservative model of replication.

Figure 51 - Analysis of DNA replication by centrifugation

Thus, copying a template strand into a complementary strand is a universal feature of both DNA Replication and Introduction/24.html">DNA Transcription into RNA. In both cases, the information contained in the template strand is preserved.

In some Viruses, single-stranded RNA molecules serve as templates for the synthesis of complementary DNA or RNA strands. However, the vast majority of RNA and DNA molecules in cells are synthesized from pre-existing duplexes.

Like RNA, DNA molecules are synthesized from deoxynucleoside 5'-triphosphates (dNTPs). Similarly to RNA Synthesis, DNA Synthesis proceeds in the 5'→3' direction because, as in RNA synthesis (Figure 8), DNA chain growth occurs via the formation of a phosphodiester bond between the 3'-oxygen of the growing chain and the α-phosphate of the dNTP.

DNA strand synthesis is catalyzed by the enzyme DNA polymerase. The substrates for DNA polymerase are four deoxyribonucleoside triphosphates: dATP, dCTP, dGTP, and dTTP.

As mentioned earlier, RNA polymerase itself locates the corresponding start site on the DNA duplex and initiates the synthesis of an RNA strand complementary to the DNA template strand.

Unlike RNA polymerase, DNA polymerase cannot independently initiate de novo chain synthesis. To do so, it requires a pre-existing DNA or RNA strand known as a primer (Figure 52).

Figure 52 - Scheme of a replication primer

Once a primer is annealed to the DNA template strand, DNA polymerase catalyzes The addition of the dNTP encoded by the template strand to the hydroxyl group at the 3'-end of the primer.

If the primer is made of RNA, the daughter strand will consist of RNA at the 5'-end and DNA at the 3'-end.