Textbook - BIOLOGICAL CHEMISTRY - Gubsky Yu.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND REALIZATION OF GENETIC INFORMATION

CHAPTER 20. MOLECULAR MECHANISMS OF DNA REPLICATION AND RNA TRANSCRIPTION

20.3. MOLECULAR MECHANISMS OF DNA REPLICATION

The General scheme of DNA Biosynthesis, originally studied using A. Kornberg's system, remains valid to this day. However, given that the DNA molecule is a double helix with antiparallel strands twisted around each other, certain geometric—topological—difficulties arise during the Replication of both prokaryotic and eukaryotic DNA molecules.

Class="center">Topological Problems of METABOLISM/36.html">DNA replication

1. Coiling and Supercoiling of the DNA Double Helix. Topoisomerases.

Native DNAs are double-helical molecules; therefore, replication of the parent molecule must be preceded by its unwinding to form a Replication fork consisting of two separated template strands (According to the semiconservative replication mechanism). Structural complexities of this process also stem from the fact that in prokaryotes (specifically E. coli), double-helical DNA molecules are circular and catenated—meaning they lack free ends and cannot be separated without breaking one of the strands.

The unwinding and Separation of the two DNA strands, which precede the synthesis of daughter strands, are accomplished through the action of specialized types of Proteins:

- topoisomerase Enzymes, which alter the number of supercoils in closed circular DNA molecules. Topoisomerases, and in particular prokaryotic DNA gyrase (from 'gyration'), introduce breaks in the polynucleotide chains of catenated DNA threads, creating the mechanical conditions necessary for their uncoiling. Topoisomerases are also present in the Chromatin apparatus of eukaryotes, where they introduce double-strand breaks in long linear chromosomal DNAs, preparing the latter for replication;

- helicase enzymes (from 'helix'), which in an ATP-dependent process unwind and separate short segments of DNA, forming replication forks—sites of sequential double-strand unwinding and synthesis of new DNA strands;

- single-stranded DNA-binding proteins (SSB proteins), which prevent their reassociation (renaturation).

The formation of a replication fork in circular double-stranded E. coli DNA molecules leads to the formation of theta structures (resembling the Greek letter 'theta'), which contain a replication fork that moves as the daughter DNA strands are synthesized:

Fig. 20.7. Circular E. coli chromosome during DNA replication. The resulting geometric Structure resembles the Greek letter θ ('theta').

(after L. Stryer, 1995; modified).

In eukaryotic DNA, which is organized as linear double-stranded molecules within nuclear chromatin, many replication forks (likely ranging from hundreds to several thousand) are formed simultaneously, facilitating the efficient replication of the entire eukaryotic chromosome. Simultaneous DNA replication at multiple points is morphologically manifested as the formation of 'replication bubbles' along the chromosome (Fig. 20.8).

Fig. 20.8. Formation of replication 'bubbles' during the doubling of eukaryotic Chromosomes (after D. Granner, 1988; modified).

Regions of the eukaryotic genome where replication forks are formed are called 'ori sites' (from 'origin'). Due to multi-origin Chromosome replication, the complete duplication of the genetic material in higher Organism Cells (such as mammals) takes about 9 hours.

2. Significance of DNA Strand Antiparallelism. Okazaki Fragments

Due to the antiparallel Nature of the two DNA strands (one 5'→3' and the other 3'→5'), the Simultaneous replication of their complementary strands would theoretically have to proceed in opposite directions (i.e., 3'→5' and 5'→3', respectively). However, as already noted, DNA polymerases can synthesize polydeoxyribonucleotide chains only in the 5'→3' direction. This contradiction was resolved through the research of Japanese scientist Reiji Okazaki, who established that the synthesis of one of the daughter DNA strands is discontinuous and occurs via short chains (1-2 thousand NUCLEOTIDES in length) that are subsequently joined together.

Fig. 20.9. Reiji Okazaki (1930-1975).

Thus, the Synthesis of the two daughter DNA strands proceeds via different mechanisms. A distinction is made between the following (Fig. 20.10):

leading strand — formed by the continuous elongation of the nucleotide chain in the 5'→3' direction;

lagging strand — formed from Okazaki fragments synthesized via DNA polymerase reactions in the 5'→3' direction.

Fig. 20.10. Scheme of DNA strand duplication according to R. Okazaki.

Stages of Synthesis of Daughter DNA Strands

1. Initiation of polydeoxyribonucleotide DNA strand synthesis, which is preceded by the formation of RNA primer strands (RNA primers) whose 3'-OH groups accept dNMPs to build new (daughter) DNA chains. These primer strands average 10 to 200 nucleotides in length. The synthesis of RNA primers is mediated by RNA polymerase enzymes known as primases.

2. Elongation of DNA Synthesis, which proceeds via different mechanisms on the leading and lagging strands.

2.1. On the leading strand, dNMP addition is carried out continuously by DNA polymerase III, extending the DNA chain from the RNA primer toward the replication fork.

2.2. On the lagging strand:

a) initially, DNA polymerase III synthesizes discrete Okazaki fragments, each beginning with its corresponding RNA primer and terminating just before THE START OF the preceding RNA primer;

b) following the formation of Okazaki fragments, DNA polymerase I uses its 5'→3' exonuclease activity to remove the RNA primers and its 5'→3' polymerase activity to replace them with DNA segments;

c) remaining gaps between individual Okazaki fragments are sealed by a specialized enzyme called DNA ligase.

The aforementioned Stages of DNA replication are illustrated in Fig. 20.11.

Fig. 20.11. Involvement of enzymes in DNA replication through the synthesis of leading and lagging strands.

Thus, coordinated action by the replisome protein complex reads information from two antiparallel parental DNA strands (oriented 5'→3' and 3'→5'), resulting in two daughter strands with corresponding antiparallel phosphodiester bond orientations (3'→5' and 5'→3', respectively). The complementary pairing of one parental and one daughter strand forms two identical DNA molecules, as predicted by the semi-conservative mechanism of replication.

This scheme of DNA replication, initially discovered in prokaryotes, also applies to Eukaryotic cells. Here too, the synthesis of the two daughter strands—leading and lagging—occurs differentially and in opposite directions. Continuous polydeoxyribonucleotide synthesis on the leading strand is catalyzed by DNA polymerase δ, whereas discontinuous synthesis on the lagging strand is mediated by polymerase α. Primases generate temporary RNA primer chains that are subsequently removed.