BIOCHEMISTRY - Textbook - Ostapchenko L. I. - 2012

Chapter 10. METABOLISM OF NUCLEIC ACIDS

10.1. Nucleases

Nucleases hydrolyze Nucleic Acids within body Tissues. Nucleases are a diverse group of Hydrolases that vary in their MECHANISM OF ACTION and Specificity, performing vital Functions at various Stages of DNA and RNA biochemical transformations. They participate in processes associated with METABOLISM/36.html">DNA Replication and Transcription, the maturation of RNA transcripts, Translation, recombination, and nucleic acid repair, as well as in protecting Cells against foreign nucleic acids.

Based on their specificity, nucleases are classified according to whether they: a) act on DNA (deoxyribonucleases), RNA (ribonucleases), or both; b) degrade single- and/or double-stranded nucleic acids; c) recognize 3'- or 5'-termini; d) are specific for purine or pyrimidine bases; or e) recognize specific nucleotide sequences.

A fundamental distinction exists between endonucleases, which target internal phosphodiester bonds within linear and circular DNA molecules to generate fragments of varying lengths, and exonucleases, which act exclusively on terminal phosphodiester bonds—at either the 5'- or 3'-end of DNA molecules—sequentially hydrolyzing phosphodiester linkages and releasing nucleoside monophosphates one by one.

Nucleases also differ in how they cleave the phosphodiester bond: some catalyze the break between the phosphate group and the 3'-OH group, generating 5'-phosphomonoester ends (Fig. 10.1, A), whereas others cleave between the phosphate group and the 5'-OH group, producing products with 3'-phosphomonoester ends (Fig. 10.1, B).

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Fig. 10.1. Two possible modes of nucleic acid phosphodiester bond Hydrolysis by nucleases

Among nucleases, Restriction Endonucleases (restriction Enzymes) deserve special attention. Unlike general nucleases, these enzymes recognize specific nucleotide sequences of 4–6 Base Pairs in double-stranded DNA molecules rather than individual nucleotide residues, thereby cleaving any given DNA into a relatively small number of fragments. Furthermore, A wide variety of restriction endonucleases exist, each recognizing a distinct sequence.

Restriction enzymes were so named because their presence in bacterial cells restricts the growth of bacterial Viruses (Bacteriophages).

Restriction enzymes are conventionally named after the bacterial species from which they are isolated. For instance, the name BamH1 indicates that the enzyme is isolated from Bacillus amyloliquefaciens. The first letter of the abbreviation corresponds to the genus (B), the next two letters represent the species (am), the letter H denotes the specific strain, and the Roman numeral indicates the chronological order of discovery of that enzyme in the given microorganism.

Restriction enzymes constitute part of the bacterial defense system that protects the host genome from foreign (primarily viral) DNA. The other component of this system consists of methylases, which specifically modify the exact same sequences recognized by restriction enzymes. Methylases catalyze The transfer of methyl groups from S-adenosylmethionine to adenine residues (forming N-6 methyladenine) or cytosine residues (forming 5-methylcytosine) within the restriction recognition site. Once methylated in this manner, The Genome is protected from self-degradation by restriction endonucleases.

Cleavage of DNA by site-specific restriction enzymes can produce "sticky" (A) or "blunt" (B) double-stranded DNA ends (Fig. 10.2). The specific DNA segments where restriction enzymes cut are referred to as restriction sites.

Fig. 10.2. Action of restriction enzymes resulting in "sticky" (A) and "blunt" (B) ends of DNA fragments

Due to their high specificity for precise nucleotide sequences, restriction endonucleases are widely used in sequencing and molecular cloning. Genome sequencing (from Latin sequentia – sequence) is a METHOD FOR DETERMINING restriction sites along a DNA molecule; establishing the relative arrangement of these sites is known as physical DNA mapping.

Molecular Gene cloning (mass replication) is a technique that enables the incorporation, isolation, and Amplification (from Latin amplificatio – enlargement) of individual genes within recipient prokaryotic or Eukaryotic cells. Cells containing the gene of interest can be utilized to produce: a) large quantities of the protein encoded by that gene, or b) large amounts of the purified gene itself.