Biochemistry and Molecular Biology - Belyasova N.A. 2002

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

The preservation of hereditary information stability, on the one hand, and the Variability of genomes, on the other hand, represent two opposing forces engaged in a constant tug-of-war. The very Organization of DNA—the molecules that encode the Genetic information of all cellular organisms—promotes the reliable storage of this data by comprising two complementary strands. If any damage occurs in one strand, the other can serve as a template to correct these distortions, a mechanism that indeed operates in numerous DNA Repair processes. Furthermore, The Double Helix enables the Replication of identical molecules, with METABOLISM/36.html">DNA replication proceeding with high fidelity (Chapter 1). Finally, many (if not all) Cells possess a defense system against The entry of foreign DNA, primarily consisting of 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 in the absence of genetic variability: despite all cellular efforts to keep their genome unchanged, it remains susceptible to alteration. The primary contributions to genome variability are made by the following processes: mutagenesis, Genetic Exchange and recombination events, as well as The activity of Mobile Genetic Elements.

The aforementioned processes, aimed at maintaining stability while altering hereditary information, are examined in this topic.

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

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

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

It is now established that the modification protecting the cellular genome and certain infecting phage DNAs is strain-specific methylation of specific nitrogenous bases within the DNA. Moreover, the enzymatic attachment of substituents to nitrogenous bases occurs after the incorporation of the corresponding NUCLEOTIDES into the DNA chains, starting from the stage of Okazaki fragment formation. Nucleotides occupying strictly defined positions in the molecule are methylated, and the enzymes catalyzing these reactions belong to a unified restriction-modification system. Restriction enzymes belonging to such a system recognize the same nucleotide sequences in DNA and perform Specific Cleavage of DNA within these (or adjacent) sequences if they are unmodified.

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

Three types of restriction endonucleases (I, II, III) are distinguished. Type I and III enzymes possess 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 perform double-stranded DNA cuts at a distance of ~25 bp from their recognition sites. Only Type II endonucleases introduce double-stranded breaks 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 identical 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. DNA cleavage by the EcoRI restrictase from Escherichia coli RY13. The STRUCTURE OF THE EcoRI restriction site is shown on the left: the dashed line represents the palindrome axis of symmetry; the vertical arrows indicate the covalent bonds between nucleotides that undergo cleavage. The DNA fragments resulting from restriction are shown on the right.

As shown in Fig. 2.1, the action of many restrictases on DNA molecules results in their staggered cleavage at unmodified sites. This mode of double-stranded DNA cleavage generates fragments with terminal redundancy, or so-called "sticky" ends. Within the "sticky" ends generated by the action of a single restrictase, The nucleotide sequences are complementary (Fig. 2.1). These functional features of Type II restrictases 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 restriction sites has been determined for the majority of them. The restriction-modification system can be viewed as a unique barrier protecting the cell against the incorporation of foreign genetic material. The Emergence of mutants lacking restriction capability opens up additional avenues for variability in these strains.

Recently, the ability of certain conjugative Plasmids and phages to overcome the restriction barriers of host cells—which they encounter during conjugation or infection—has been discovered. This phenomenon has been named anti-restriction. Genes designated ard, which determine the structure of anti-restriction Proteins, have been discovered in the genomes of plasmids and phages. These proteins inhibit enzymes belonging to the Type I restriction-modification system.