Biochemistry and Molecular Biology - Belyasova N.A. 2002

Molecular Foundations and Mechanisms of Heredity
Maintenance of Stability and Variability of Genomes
The Phenomenon of DNA Restriction-Modification

Preserving the constancy of hereditary information, on the one hand, and genome Variability, on the other, represent two opposing forces locked in a constant interplay. The very Organization of DNA—the molecules encoding the Genetic information of all cellular organisms—promotes the reliable storage of this data by utilizing two complementary strands. If one strand sustains any damage, the other can serve as a template to correct these distortions, a mechanism actively employed in numerous DNA Repair processes. Furthermore, The Double Helix enables the Replication of similar molecules, ensuring that METABOLISM/36.html">DNA replication proceeds with high fidelity (Chapter 1). Finally, many (if not all) Cells possess a defense system against the intrusion of foreign DNA, primarily relying on a set of Nucleases—Enzymes capable of degrading Nucleic Acids. Among these nucleases, restriction enzymes (Restriction Endonucleases) are of particular importance.

However, evolution would be impossible without genetic variability: despite all cellular efforts to keep their genomes unchanged, genomes are nonetheless subject to alteration. The primary contributors to genome variability are mutagenesis, Genetic Exchange and recombination events, and The activity of Mobile Genetic Elements.

These processes, directed toward maintaining the constancy and driving the evolution of hereditary information, are examined in this topic.

The restriction-modification system comprises two classes of enzymes: one Class modifies DNA molecules present within The Cell, while the other cleaves foreign DNA molecules (or unmodified host DNA) at the same specific sites.

The restriction-modification phenomenon was first discovered by S. Luria in the 1950s during experiments involving the infection of E. coli Bacteria with bacteriophage lambda. It was found that Bacteriophages propagated on one strain of E. coli infected cells of certain other strains with low efficiency. The reverse was also observed: phages produced during this low-productivity lytic cycle infected Cells of the original strain equally poorly. It was suggested,

and later confirmed, that phage DNA undergoes modification within E. coli bacteria, which protects it against host restriction enzymes, but not against similar enzymes in other strains.

It is now known that the modification protecting the cell genome and certain infecting phage genomes is the strain-specific methylation of specific nitrogenous bases in DNA. Furthermore, the enzymatic attachment of substituents to nitrogenous bases occurs after the incorporation of the corresponding NUCLEOTIDES into the DNA chains, beginning at the stage of Okazaki fragment formation. Nucleotides occupying strictly defined positions within the molecule are methylated, and the enzymes catalyzing these reactions belong to a unified restriction-modification system. The restriction enzymes within such a system recognize the same nucleotide sequences in DNA and effect Specific Cleavage at these (or adjacent) sequences if they remain unmodified.

The Structure of restriction-modification sites has been deciphered for more than 200 restriction enzymes.

Three types of restriction endonucleases (I, II, and III) are distinguished. Type I and III enzymes exhibit both nuclease and methylating activities. Specifically, Type I endonucleases recognize a specific sequence in DNA and cleave double-stranded DNA at variable (non-fixed) distances from the recognition sites. Type III endonucleases introduce double-stranded DNA breaks at a distance of ~25 bp from their recognition sites. Only Type II endonucleases make double-stranded cuts in DNA at specific phosphodiester bonds either within the recognition site itself or at a short, well-defined distance from it. Enzymes of this type lack methylating activity. Some Type II endonucleases recognize specific groups of four nucleotides, while others recognize hexanucleotide sequences; a common feature among them is a palindromic structure (Fig. 2.1). In this case, the same sequence is located in both strands in opposite directions, symmetrically relative to the axis of Symmetry in the middle of the palindrome.

Fig. 2.1. Cleavage of DNA by the EcoR1 restriction enzyme from Escherichia coli RY13. Left: STRUCTURE OF THE EcoR1 restriction site, where the dashed line indicates the axis of symmetry of the palindrome, and vertical arrows denote the covalent bonds between nucleotides that undergo cleavage. Right: DNA fragments generated As a result of restriction

As shown in Fig. 2.1, the action of many restriction enzymes on DNA molecules results in staggered cuts at unmodified sites. This mode of double-stranded DNA cleavage produces fragments with terminal redundancy, commonly referred to as "sticky" ends. The nucleotide sequences within "sticky" ends generated by the action of a single restriction enzyme are complementary (Fig. 2.1). These Functional Characteristics of Type II restriction enzymes make them indispensable tools in Introduction/32.html">Genetic Engineering: they act as molecular scissors used to fragment DNA in vitro.

Currently, restriction enzymes have been isolated from more than 400 bacterial strains, and the structure of their restriction sites has been determined for the majority of them. The restriction-modification system can be regarded as a peculiar barrier protecting the cell against the incorporation of foreign genetic material. The Emergence of mutants lacking restriction capability opens up additional opportunities for variability in these strains.

Recently, the ability of certain conjugative Plasmids and phages to overcome the restriction barriers of host cells—which they enter during conjugation or infection—has been discovered. This phenomenon has been termed antirestriction. The genomes of these plasmids and phages have been found to contain ard genes that determine the structure of antirestriction Proteins. These proteins inhibit enzymes belonging to the Type I restriction-modification system.