Textbook - BIOLOGICAL CHEMISTRY - Hubsky Y.I. - 2000

Chapter IV. MOLECULAR MECHANISMS OF HEREDITY AND THE EXPRESSION OF GENETIC INFORMATION

CHAPTER 20. MOLECULAR MECHANISMS OF DNA REPLICATION AND RNA TRANSCRIPTION

20.2. DNA REPLICATION ENZYMES IN PROKARYOTES AND EUKARYOTES

Class="center">Research by A. Kornberg (Discovery of DNA Polymerase I)

General scheme of DNA Biosynthesis in A. Kornberg's System

A. Kornberg et al. (Kornberg Arthur, 1956) incubated E. coli extracts containing Enzymes and a certain amount of DNA with a mixture of deoxynucleoside triphosphates (dNTPs), namely: dATP, dTTP, dGTP, and dCTP.

It was established that under these conditions, a certain amount of new DNA (more precisely, polydeoxyribonucleotide) is synthesized, which incorporates deoxynucleoside monophosphates (dNMPs) derived from the dNTPs used for biosynthesis. In this process, the new DNA strand was formed by attaching a dNMP to the end of a pre-existing DNA strand.

The reaction equation in its simplest form is as follows:

The enzyme that catalyzed this reaction was named DNA polymerase I, and its study helped clarify several fundamental features of DNA biosynthesis in both PROKARYOTES AND EUKARYOTES. Although it turned out not to be the primary enzyme in METABOLISM/36.html">DNA Replication, it performs certain auxiliary Functions in this process.

The Requirement for Template (Pre-existing) DNA in Kornberg's System

DNA polymerase I, which mediated The formation of 3'-5' phosphodiester bonds in Kornberg's system, could function only in the presence of pre-existing DNA. The pre-existing DNA required for the Formation of the new polydeoxyribonucleotide serves two functions (Fig. 20.6). It acts as:

(1) a primer for the elongation of the polydeoxyribonucleotide chain;

(2) a template that determines (programs) the sequence in which new NUCLEOTIDES are incorporated into the synthesized chain.

Fig. 20.6. Scheme of new DNA strand synthesis in the presence of primer and template strands.

It was later established that DNA polymerase I (the "Kornberg enzyme") is not the primary enzyme responsible for DNA Introduction/23.html">Replication in Prokaryotes. Nevertheless, certain features of this synthesis proved applicable to the functioning of other polymerases as well.

KEY FEATURES OF DNA Synthesis in the DNA polymerase system:

- DNA polymerase cannot synthesize a completely new DNA strand from scratch; it is only capable of adding dNMPs to an already existing strand;

- Synthesis of the new DNA strand proceeds in the 5'-3' direction, meaning DNA polymerase sequentially adds nucleotides (dNMPs from available dNTPs) to the 3'-end of one of the DNA strands (the "primer" strand);

- a template strand is also required for the synthesis of a new DNA strand; nucleotides are joined to the 3'-end of the "primer" strand in accordance with The nucleotide sequence of the "template" strand (i.e., according to THE PRINCIPLE OF complementarity).

The Mechanism of DNA chain elongation involves the formation of new 3'-5' phosphodiester bonds synthesized in the 5'→3' direction. This process occurs via a nucleophilic attack by the free 3'-hydroxyl group of the terminal nucleotide of the pre-existing chain on the α-phosphate of the incoming dNTP:

Enzymes of DNA Biosynthesis in Prokaryotes

1) DNA polymerase I — a protein with a molecular mass of 103 kD.

Biochemical functions:

5'→3' — polymerase activity (discussed above);

5'→3' — exonuclease activity (i.e., The ability to remove nucleotides upstream of the synthesis direction);

3'→5' — exonuclease activity ("proofreading" activity, or the ability to remove an already incorporated nucleotide if it was added erroneously, meaning it is not complementary to the template strand);

2) DNA polymerase II (a protein with a molecular mass of 120 kD) is not the primary enzyme in prokaryotic DNA replication; current understanding suggests that the predominant function of this enzyme is involvement in DNA Repair;

3) DNA polymerase III is the principal enzyme that carries out DNA elongation in E. coli.

In terms of its molecular Structure, DNA polymerase III is an asymmetric dimer with a molecular mass of approximately 900 kD. It is a multimeric enzyme whose entire molecule (holoenzyme) contains 10 types of subunits — polypeptide chains.

The enzyme exhibits 5'→3' polymerase, 3'→5' exonuclease, and ATPase activities. DNA polymerase III is a highly processive enzyme capable of catalyzing the linkage of many thousands of mononucleotides into a chain without dissociating from the template (unlike the 15–20 nucleotides synthesized by DNA polymerase I). The rate of the polymerization reaction is approximately 1000 nucleotides per second.

ATPase activity is a prerequisite for the formation of the DNA polymerase holoenzyme-template complex: first, ATP interacts with the β-subunit of the holoenzyme, and then the activated holoenzyme binds to the DNA template strand, which is accompanied by ATP Hydrolysis.

Eukaryotic DNA Biosynthesis Enzymes

Several types of DNA polymerases have been found in Eukaryotic Cells (Table 20.1).

Table 20.1. Eukaryotic DNA Polymerases

The Key Enzymes playing a major role in eukaryotic DNA replication are DNA polymerases α and δ, which are highly processive enzymes with 5'→3' polymerase activity. Polymerases β and ε are involved in nuclear DNA repair, whereas γ-polymerase is responsible for Mitochondrial DNA replication.

The levels of DNA polymerase α increase markedly during the S-phase of the Cell Cycle, when active DNA synthesis takes place. The activity of DNA polymerases α, δ, and ε is specifically inhibited by aphidicolin, a diterpenoid derived from Fungi that acts as a potent antitumor antibiotic.