BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 10. NUCLEIC ACID METABOLISM

10.6. DNA Biosynthesis (Replication)

10.6.1. Replication Enzymes

Despite the simplicity of its core principle, the Replication process is intricately organized and requires the participation of numerous Proteins and Enzymes. Interestingly, these enzymes, like all other proteins, are encoded within the DNA nucleotide sequence. This creates a feedback loop: DNA directs the Synthesis of the proteins that subsequently replicate the DNA.

The primary enzymes involved in METABOLISM/36.html">DNA replication are DNA-dependent DNA polymerases. All DNA polymerases from both prokaryotic and eukaryotic organisms share the same fundamental synthesizing activity—they add NUCLEOTIDES to the OH group at the 3'-end of a DNA strand, causing it to grow in the 5'→3' direction.

For enzymes to carry out the 5'→3' polymerase reaction, the following conditions are necessary:

✵ a template, which guides the synthesis of the complementary polynucleotide chain; within the Replication fork, a single-stranded region of DNA serves as this template;

✵ a primer (from the English 'primer'); DNA polymerases are incapable of forming internucleotide bonds between two free nucleotides, and can only extend a pre-existing oligonucleotide; consequently, DNA Synthesis begins only after a small oligoribonucleotide primer attaches to the DNA template;

✵ substrates; the substrates for DNA chain synthesis are four deoxyribonucleotide-5'-triphosphates: dATP, dGTP, dCTP, and dTTP.

Both prokaryotic and Eukaryotic Cells possess several different DNA polymerases. The best-studied are the DNA polymerases of E. coli. Three distinct types of DNA polymerases—I, II, and III—have been identified in these Bacteria, differing primarily in their catalytic rates and nuclease activities.

DNA polymerase I exhibits the following activities:

✵ polymerase activity—The addition of deoxyribonucleotides, complementary to the template strand, to the free 3'-OH group of the primer in the 5'- to 3'-direction relative to the newly synthesized DNA molecule;

✵ exonuclease activity—the Hydrolysis of phosphodiester bonds (removal of nucleotides) within a single DNA strand or at an unpaired end of double-stranded DNA, starting from either the 3'-end (3'→5') or the 5'-end (5'→3') of the chain. The 3'→5' exonuclease activity provides proofreading by monitoring the addition of each nucleotide and removing incorrect ones from the growing end of the chain, whereas the 5'→3' exonuclease activity is used to remove the ribonucleotides of Okazaki fragments, a necessary prerequisite for ensuring the continuity of the lagging strand.

The ability of DNA polymerase I to extend the 3'-end of a strand at a nick in double-stranded DNA and to remove nucleotides from the 5'-end of the same nick enables the Repair of DNA damage. This process is known as nick Translation.

DNA polymerase II exhibits polymerase activity at a slower rate than DNA polymerase I and lacks 5'→3' exonuclease activity. While this enzyme is considered non-essential for DNA replication, it is capable of filling gaps

between DNA fragments paired with the template strand. Thus, the presence of both polymerase and 3'→5' exonuclease activities suggests that DNA polymerase II participates in DNA Repair.

DNA polymerase III is the primary enzyme responsible for E. coli DNA replication. It serves as a core component of the multienzyme complex that initiates replication fork formation at origins of replication, participates in leading strand elongation, and extends RNA primers to form Okazaki fragments. In addition to its polymerase activity, DNA polymerase III possesses 3'→5' exonuclease activity. The latter is activated when an error occurs and an "incorrect" nucleotide is incorporated into the growing chain. In such cases, the enzyme recognizes the mismatch, removes the terminal nucleotide from the growing 3'-end, and subsequently resumes its polymerase activity. This mechanism ensures continuous proofreading of the addition of each nucleotide by the system.

A characteristic feature of DNA polymerase III is its exceptionally high processivity in DNA synthesis—referring to the number of nucleotides added by the enzyme to the growing chain between successive dissociation events from the template.

Six types of DNA polymerases have been identified in eukaryotic cells—α, β, γ, δ, ε, and ζ. They differ in subunit composition, molecular weight, association with various accessory proteins that accelerate DNA Biosynthesis, and functional roles. DNA polymerases β, δ, and ε are unable to initiate daughter DNA strands because they lack affinity for single-stranded DNA. Replication is initiated by DNA polymerase α, which synthesizes RNA primers ranging from 10 to 60 nucleotides in length, while ε-polymerase is responsible for synthesizing the lagging strand (Okazaki fragments). The function of DNA polymerase β involves the stepwise removal of the RNA primer from the 5'-end, followed by the addition of deoxyribonucleotides to the 3'-end of the preceding Okazaki fragment in an amount equal to the removed primer, thereby filling the gap left by the excised ribonucleotides. DNA polymerase δ is responsible for leading strand synthesis and also exhibits proofreading 3'→5' exonuclease activity. DNA polymerase ζ displays relatively high 3'→5' exonuclease and low polymerase activities, indicating an auxiliary proofreading role during DNA replication. DNA polymerase γ is a mitochondrial enzyme responsible for the synthesis of Mitochondrial DNA.

The Active Site of all DNA polymerases (as well as RNA polymerases) contains a Zn²⁺ ion (an enzyme cofactor). The interaction of DNA polymerases with substrates also requires the presence of Mg²⁺ ions, which polarize nucleotides by forming complexes with them and enhancing their reactivity.

As a result of DNA polymerase action, each newly synthesized lagging strand consists of discrete fragments that are subsequently joined into a continuous strand. This ligation is mediated by another essential replication enzyme, DNA ligase. This enzyme catalyzes The formation of a covalent phosphodiester bond between the 5'-phosphoryl group of one fragment and the 3'-hydroxyl group of the deoxyribose on the neighboring fragment. The reaction requires high-energy Cofactors: E. coli DNA ligase requires NAD+, whereas eukaryotic DNA ligase requires ATP. Notably, the enzyme exhibits activity toward single-stranded DNA fragments embedded within double-stranded DNA.

DNA ligases also participate in the joining (ligation) of DNA strands during other template-directed processes, specifically DNA repair and recombination.

Topological rearrangements of DNA. The rearrangements that occur during replication may be associated with reactions that induce structural changes within DNA molecules.

First and foremost, during replication, double-stranded DNA must unwind and separate into individual strands so that each can function as a template. Such topological rearrangements of DNA are driven by specialized proteins. For instance, the family of Cleavage-rejoining enzymes—DNA topoisomerases—participates in regulating DNA Supercoiling. DNA molecules that differ in their degree of supercoiling are referred to as topological isomers (topoisomerases), which is THE ORIGIN OF these enzymes' name.

Topoisomerases alter the degree and type of DNA supercoiling. Some topoisomerases relax supercoiled DNA, whereas others induce tension and promote the formation of supercoils. Two Types of topoisomerases have been identified in various organisms:

Type I topoisomerases can transiently cleave one of the two DNA strands. The proximal end of the DNA forms a covalent bond with the enzyme, while the distal region (spanning from the unwinding point to the break) is free to rotate around the intact strand. As a result, the num

ber of supercoils in the supercoiled DNA decreases, converting it into a relaxed form. The covalent linkage between the enzyme and DNA preserves a significant amount of energy by retaining the energy of the cleaved phosphodiester bond. Release of the enzyme leads to rapid ligation of the broken DNA ends without requiring additional energy.

Type II topoisomerases can introduce transient double-strand breaks in both complementary DNA strands, with one of the ends being covalently held by the topoisomerase. In this state, the degree of DNA supercoiling is altered before the broken ends are rejoined. Type II enzymes are active in the presence of ATP, the hydrolysis of which is required to drive Conformational Changes in the protein.

Hydrogen Bonds within the double-stranded DNA molecule are disrupted by DNA helicases (from the Greek helix), which generate the mechanical conditions necessary to separate the strands and form the replication fork. These enzymes utilize The energy released during ATP hydrolysis to unwind the DNA strands.

Unwound regions of DNA are maintained in a single-stranded state by tetrameric SSB proteins (single-stranded DNA-binding proteins). These proteins bind with high affinity to single-stranded DNA along the entire length of the separated strands, thereby preventing their complementary re-annealing. Furthermore, SSB proteins bind cooperatively to DNA—meaning that through Protein-Structure/156.html">Protein Interactions, the tetramers pack closely together along the single-stranded DNA, keeping the nitrogenous bases accessible for complementary pairing during replication.