Fundamentals of Biochemistry - A. A. Anisimov 1986

Nucleic Acids
Enzymes for the Synthesis and Transformation of Nucleic Acids

4.6.1. DNA Polymerases. Bacterial Cells contain several DNA polymerases. DNA polymerase I was the first to be discovered in E. coli extracts by A. Kornberg's research group (1956). This enzyme catalyzes the polymerization of NUCLEOTIDES on a single-stranded template. A DNA primer is required to initiate synthesis. Polymerization proceeds via The addition of mononucleotides to the 3'-OH group of the DNA primer. The template dictates the enzyme's Selection of nucleotides according to base-pairing rules: A pairs with T, and G with C. Chain elongation proceeds in the 5'→3' direction. DNA polymerase I specifically requires deoxynucleoside 5'-triphosphates; 5'-diphosphates and 5'-monophosphates are inactive, as are ribonucleoside 5'-triphosphates. Mg²⁺ ions are required for the reaction.

During METABOLISM/36.html">DNA Replication, the synthetic activity of DNA polymerase I plays an auxiliary role—the enzyme "fills in" the gaps that form between DNA fragments following the removal of RNA primers (see Section 4.7.2). DNA polymerase I also performs a proofreading function, i.e., it excises incorrectly incorporated nucleotides. In this capacity, the enzyme exhibits 3'→5' exonuclease activity.

DNA polymerase I plays a vital role in DNA Repair, which involves correcting damaged Regions of the DNA molecule. Unlike other DNA polymerases, DNA polymerase I can synthesize DNA on a template containing multiple nicks. This capability is attributed to its intrinsic 5'→3' exonuclease activity: DNA polymerase I excises a series of nucleotides, enlarging the gap to a size where it can serve as a "starting platform" for synthesis.

DNA polymerase I also plays a crucial role in excising thymine dimers induced by ultraviolet irradiation of cells and in filling the resulting gaps. By simultaneously catalyzing nucleotide incorporation at the 3' end and nucleotide removal at the 5' end, DNA polymerase I drives the progression of a single-strand break along the DNA molecule. This translocation of a nick, or "nick Translation," presumably also occurs during the repair of UV-induced cellular damage. Thus, The Physiological Role of DNA polymerase I in The Cell is immense.

E. coli Cells also contain DNA polymerase II. Its function is primarily restricted to the Repair of Damaged regions within the DNA molecule. If a DNA gap spans 2 to 100 nucleotides, DNA polymerase II fills it in by incorporating nucleotides starting from the 3'-OH end of the gap. For larger gaps, DNA polymerase II is either unable to mediate repair or does so only partially. In the absence of DNA polymerase I, DNA polymerase II can complete the synthesis of DNA fragments generated during DNA replication.

In 1972, T. Kornberg and coworkers isolated DNA polymerase III from E. coli cells. It plays the primary role in DNA replication. Like other polymerases, the enzyme catalyzes polymerization exclusively in the 5'→3' direction. In vitro, the template is a double-stranded DNA molecule containing numerous short gaps and, consequently, free 3'-OH ends. However, the active complex of DNA polymerase III with two specific Proteins, in the presence of co-polymerase III, operates only on a long template. The catalytic activity of DNA polymerase III is 15 times higher than that of DNA polymerase I and 300 times higher than that of DNA polymerase II.

Eukaryotic cells, much like E. coli and other prokaryotes, harbor multiple DNA polymerases. Unlike their prokaryotic counterparts, eukaryotic DNA polymerases lack exonuclease activity and therefore cannot perform proofreading Functions. The predominant enzyme is designated as a-DNA polymerase. It is particularly abundant in rapidly proliferating cells and is responsible for the replication of nuclear DNA. The second enzyme, ß-DNA polymerase, is involved in nuclear DNA repair and is absent in lower eukaryotes (such as Yeast and lower plants). The third, y-DNA polymerase, has been localized to the Mitochondria. It differs from the a- and ß-Enzymes in its physicochemical properties and presumably catalyzes Mitochondrial DNA replication.

4.6.2. DNA-Dependent RNA Polymerase. All cellular RNA types—messenger, ribosomal, and Transfer RNA—are synthesized on a DNA template through the action of DNA-dependent RNA polymerase. The sequence of deoxyribonucleotides in the DNA strand is transcribed, effectively translated into a different molecular language (hence the term Transcription, see Section 4.8.1), into a ribonucleotide sequence in RNA. In simplified terms, the RNA polymerase reaction can be represented as follows:

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Mononucleotides are added one by one to the 3'-hydroxyl end of the RNA chain According to the base-pairing principle; the chain elongates in the 5'→3' direction, with nucleotides joined by 3'—5' phosphodiester bonds. The reaction requires all four ribonucleoside triphosphates and either Mg or Mn ions. Although double-stranded DNA typically serves as the template, only one of its strands—the one carrying the anticodon sequences—is transcribed, ensuring that the resulting mRNA contains codons. As a rule, only a specific region of DNA encoding a single enzyme or a group of enzymes is copied.

RNA polymerases are found in animal, plant, and bacterial cells. The RNA polymerase of E. coli and certain Bacteriophages has been the most thoroughly characterized. In E. coli, RNA polymerase has a complex architecture and a high molecular weight (M = 480,000). It consists of the following subunits: two a—(39,000), ß—(155,000), ß'—(165,000), and o—(95,000). The complex comprising only the a₂-, ß-, and ß'-subunits is termed the core enzyme. It catalyzes RNA Synthesis, but in the absence of the σ-subunit, it does so nonspecifically; the σ-factor stabilizes the enzyme-DNA complex at specific promoter sites. Binding of σ to the core enzyme yields the complete holoenzyme.

In addition to the transcriptional RNA polymerase, E. coli possesses another RNA polymerase whose function is to synthesize short RNA primers required for the initiation of replication and DNA fragment synthesis (see Section 4.7.2). Small phage RNA polymerases have also been identified in Prokaryotic Cells; these utilize phage DNA templates to synthesize mRNAs that encode phage proteins.

All eukaryotes, ranging from yeast to mammals, possess a system of multiple RNA polymerases. Eukaryotic RNA polymerases have been isolated from calf Thymus, human cell cultures, rat Liver, and yeast. Three distinct enzymes, which are high-molecular-weight Structure/178.html">Protein Complexes containing complex sets of subunits, have been identified in each of these sources. All of them are nuclear enzymes.

RNA polymerase I is localized in the nucleolus and catalyzes the synthesis of Ribosomal RNAs. This enzyme exists in two forms: one consists of 5–6 subunits, while the other contains one fewer subunit. RNA polymerase II is present in the nucleoplasm and transcribes all protein-coding eukaryotic genes as well as major animal virus genes; it consists of five subunits. RNA polymerase III is also localized in the nucleoplasm and is believed to be responsible for the synthesis of 5S rRNA and tRNAs; it consists of 10 or more subunits. The molecular weights of the subunits across these RNA polymerases vary over a wide range.

Eukaryotic mitochondria possess an independent apparatus for template-directed synthesis. The simplest known RNA polymerase—a monomer with M ≈ 64,000—has been isolated from them.

4.6.3. Reverse Transcriptase. Reverse transcriptase, also known as convertase or RNA-dependent DNA polymerase, has attracted extraordinary scientific interest. It was discovered within RNA-containing Oncogenic Viruses such as Rous Sarcoma virus, avian myeloblastosis virus, Rauscher leukemia virus, and in several human tumors. Prior to its discovery, it was dogma that Genetic information flows exclusively from DNA to RNA (transcription). Because this enzyme synthesizes DNA from an RNA template and transfers genetic information from RNA to DNA, it was named reverse transcriptase or convertase. Reverse transcriptase exhibits three distinct enzymatic activities.

The first of these is RNA-dependent DNA polymerase activity. It directs the synthesis of single-stranded complementary DNA (cDNA) using an RNA template and requires a primer—ranging from 4 to 200 nucleotides in length and composed of either ribonucleotides or deoxyribonucleotides—that must possess a free 3'-OH group.

The second activity is DNA-dependent DNA polymerase activity, which drives the Synthesis of the second DNA strand (anti-cDNA) and is responsible for converting single-stranded DNA into a double-stranded form on a DNA template. The third is RNase H activity, which specifically hydrolyzes the RNA moiety within an RNA-DNA hybrid. Initially, reverse transcriptase synthesizes a complementary DNA strand on the RNA template, yielding a double-stranded RNA-DNA hybrid molecule. The original RNA template is then degraded. Using the remaining DNA strand as a template, reverse transcriptase synthesizes the second DNA strand, resulting in a final double-stranded DNA molecule.

The double-stranded viral DNA generated in this manner can integrate into The Genome (chromosome) of the host cell. Subsequent events lead either to the Propagation of the oncornavirus (RNA tumor virus) or to tumorigenesis. The Gene encoding reverse transcriptase is embedded within the oncornavirus genome.

Characterization of reverse transcriptase has revealed that it lacks template and primer Specificity. This property has enabled its widespread application in Introduction/32.html">Genetic Engineering for gene synthesis, the analysis of various regions of viral genomes, the quantification of genes in eukaryotic cells, the determination of their primary structures, and The Study of RNA Processing.

4.6.4. DNA Ligases. Single-strand breaks in DNA are repaired by enzymes known as DNA ligases. They catalyze The formation of a phosphodiester bond between a 3'-OH group and a 5'-phosphoryl group of DNA fragments situated within a distance of a single nucleotide gap. The synthesis is coupled with the Cleavage of a pyrophosphate bond in NAD⁺ (in E. coli and other prokaryotes) or ATP (in eukaryotes). This seals the nicks to form covalently closed DNA strands.

During this process, NMN (nicotinamide mononucleotide) is cleaved from NAD⁺, while H₄P₂O₇ is released from ATP. The AMP generated in both cases is first transferred to the enzyme and subsequently to the 5' end of the DNA strand to be joined, activating it before being released upon completion of the ligation reaction. Ligases play a crucial role in NUCLEIC ACID METABOLISM, participating in DNA replication, repair, and recombination. In genetic engineering, ligases are widely used for "splicing" DNA molecules.

4.6.5. Replicase. The replication of RNA in RNA-containing Viruses is mediated by an RNA-dependent RNA polymerase (RNA replicase). The synthesis of this enzyme within a virus-infected cell is induced by viral RNA. The replicase utilizes nucleoside triphosphates to synthesize single-stranded viral RNA. In The First stage of synthesis, the enzyme uses the viral RNA template to produce a complementary RNA of opposite polarity (the replicative form). In the second stage, this strand serves as a template for the synthesis of numerous new viral RNA strands. Both stages of synthesis are catalyzed by the same enzyme, although each requires distinct protein accessory factors.

4.6.6. Polynucleotide Phosphorylase. Polynucleotide Phosphorylase catalyzes the synthesis of RNA from ribonucleoside diphosphates: RNA_n + NDP ⇄ RNA_n+1 + P_i. The reaction requires An RNA primer chain with a free 3'-OH group to which mononucleotides are added. The COMPOSITION OF THE product is determined entirely by The ratio of ribonucleoside diphosphates in the initial reaction mixture, making it possible to synthesize polymers with predefined nucleotide compositions in vitro. Consequently, polynucleotide phosphorylase was instrumental in constructing artificial templates for deciphering The Genetic Code (see Section 5.3.5). Because the reaction catalyzed by polynucleotide phosphorylase is reversible, it is believed to mediate the degradation of short-lived mRNAs within the cell.

4.6.7. DNA-(cytosine-5)-methyltransferases (DNA methyltransferases). It is known that, alongside the principal nitrogenous bases, DNA contains minor components—methylated, glucosylated, and otherwise modified bases. Together with the Features of the Primary Structure, these components ensure the species specificity of DNA. The modification of standard nitrogenous bases occurs after the DNA chains have already been synthesized. For example, the 6-amino group of adenine and the C-5 of cytosine undergo methylation. A methyl group is transferred from S-adenosylmethionine to the adenine or cytosine residue within the DNA strand. This process is catalyzed by the enzyme DNA methyltransferase. Methylation takes place at strictly defined sites on the DNA molecule where specific nucleotide sequences are located. Methylation protects DNA from the cleavage activity of Restriction Endonucleases.

4.6.8. Nucleases. Enzymes that cleave phosphodiester bonds in nucleic acid molecules are termed nucleases. The cleavage of a phosphodiester bond can occur in two ways: in one case, products phosphorylated at the 5'-end are formed, and in the other, at the 3'-end. A distinction is made between endonucleases and exonucleases. Endonucleases hydrolyze phosphodiester bonds within the molecule simultaneously at many sites, yielding fragments of varying sizes. Exonucleases remove nucleotides one by one from one of the ends of the polynucleotide chain. Depending on their substrate specificity, nucleases are divided into two groups: ribonucleases (RNases) and deoxyribonucleases (DNases).

The cellular functions of RNases are diverse. For instance, RNase I cleaves various types of RNA into mononucleotides, which are subsequently utilized for the synthesis of new RNAs. RNases II, III, IV, and P participate in the processing of mRNA, rRNA, and tRNA, which are initially produced as precursor molecules. RNase specifically cleaves RNA within DNA-RNA hybrid complexes. It may be involved in the cleavage of RNA primers during DNA replication, as well as in the degradation of RNA during the action of reverse transcriptase.

The group of DNases is likewise represented by a variety of enzymes. Endo-DNases include DNase I and DNase II. DNase I has been isolated from the Pancreas; it cleaves internal phosphodiester bonds in one of the DNA strands, yielding oligonucleotides with a 5'-terminal phosphoryl group. DNase II is found in the Spleen and thymus. It catalyzes the cleavage of both DNA strands, resulting in the formation of 3'-phosphooligonucleotides. Several DNases have been isolated from Bacteria.

Great research interest has been attracted to DNases that cleave DNA at strictly defined sites harboring specific nucleotide sequences, typically palindromic and 4–6 nucleotides in length. These enzymes are called restriction enzymes (DNA Restriction enzymes, or restriction endonucleases). A restriction enzyme and a methylase "recognize" the same multinucleotide sequence in DNA. The restriction enzyme cleaves double-stranded DNA provided that the bases of the recognition sequence are unmethylated in both strands.

Using restriction enzymes, bacteria can destroy foreign DNA that has entered the cell via conjugation or viral infection. Thus, restriction enzymes protect the bacterial cell from invading foreign DNA. To remain uncleaved within the cell, foreign DNA must be methylated in a specific manner. Restriction Enzymes exhibit specificity toward: 1) the recognition sequence, 2) the restriction (cleavage) site, and 3) the methylation of the bases within the recognition sequence.

The names of restriction enzymes consist of three letters: the first letter indicates the genus of the source microorganism, and the next two letters designate the species. If a restriction enzyme is encoded by a plasmid or phage gene, the symbol of this non-chromosomal element is also indicated, e.g., EcoR. When a strain contains multiple restriction enzymes, they are denoted by Roman numerals: Hind I, Hind II, Hind III. If different strains of bacteria of the same species harbor different restriction enzymes, a strain number or letter is appended to the three-letter designation: Eco B, Eco K.

Restriction enzymes are divided into three types. Type I and Type III enzymes exhibit two activities: restriction and methylation. Both types of enzymes recognize a specific unmethylated nucleotide sequence in DNA. Type I restriction enzymes (e.g., Eco K, Eco B) are characterized by relatively low specificity for cleavage sites. Type III enzymes (Eco PI, Hind III) "cut" DNA at specific sites, but do not drive the cleavage to completion because they simultaneously catalyze methylation, which prevents further cleavage. Enzymes of both types possess a high molecular weight (200,000–400,000).

Type II restriction enzymes consist of two separate proteins: the restriction enzyme proper and the methylase. These enzymes "recognize" a specific sequence in DNA, which is typically a palindrome of 4 or 6 Base Pairs. The restriction enzyme, which has a relatively low molecular weight (approximately 60,000), "cuts" either this sequence or a sequence located immediately adjacent to it. For example, EcoRI endonuclease recognizes a hexanucleotide sequence and cleaves it at the sites indicated by arrows (asterisks indicate methylation sites): The cleavage sites are palindromic. EcoRII endonuclease cleaves the two DNA strands at different sites; therefore, the cleavage products possess "sticky" ends.

The endonuclease Hind II (a restriction enzyme from Hemophilus influenzae) cleaves DNA to produce "blunt" ends at the following sites:

Methylases modify DNA bases at these exact same specific sites.

The most distinct difference between Type I and Type III enzymes, on the one hand, and Type II enzymes, on the other, lies in The Nature of their cleavage products. The action of Type I restriction enzymes on DNA yields heterogeneous products, whereas Type III yields products of incomplete substrate cleavage. Only Type II restriction enzymes specifically and completely cleave DNA into fragments whose lengths correspond to the distances between the recognition sequences. Therefore, Type II restriction enzymes are widely used in genetic engineering. Because restriction enzymes cleave DNA strands at sites with strictly specific nucleotide sequences, they are employed in experiments aimed at determining the Primary structure of DNA.

4.6.9. Topoisomerases. DNA topoisomerases are enzymes that alter the supercoiling number in covalently closed circular DNA. Topoisomerases that act on both single-stranded and double-stranded DNA molecules are known. Among them, enzymes have been discovered that link two or more DNA molecules into catenanes (interlocked rings). Topoisomerase I breaks one of the strands of a circular supercoiled DNA, allowing the strands to unwind and reducing the supercoil number, after which the same enzyme reseals the break.

Belonging to the same class of enzymes is DNA gyrase (topoisomerase II). Gyrase converts relaxed, non-supercoiled closed circular DNA into a supercoil—that is, it exhibits an action opposite to that of topoisomerase I. This reaction requires a significant expenditure of ATP. If gyrase is inactivated, critical cellular processes, particularly DNA replication, are disrupted.