Structure and function of type II restriction endonucleases

Abstract

More than 3000 type II restriction endonucleases have been discovered. They recognize short, usually palindromic, sequences of 4-8 bp and, in the presence of Mg(2+), cleave the DNA within or in close proximity to the recognition sequence. The orthodox type II enzymes are homodimers which recognize palindromic sites. Depending on particular features subtypes are classified. All structures of restriction enzymes show a common structural core comprising four beta-strands and one alpha-helix. Furthermore, two families of enzymes can be distinguished which are structurally very similar (EcoRI-like enzymes and EcoRV-like enzymes). Like other DNA binding proteins, restriction enzymes are capable of non-specific DNA binding, which is the prerequisite for efficient target site location by facilitated diffusion. Non-specific binding usually does not involve interactions with the bases but only with the DNA backbone. In contrast, specific binding is characterized by an intimate interplay between direct (interaction with the bases) and indirect (interaction with the backbone) readout. Typically approximately 15-20 hydrogen bonds are formed between a dimeric restriction enzyme and the bases of the recognition sequence, in addition to numerous van der Waals contacts to the bases and hydrogen bonds to the backbone, which may also be water mediated. The recognition process triggers large conformational changes of the enzyme and the DNA, which lead to the activation of the catalytic centers. In many restriction enzymes the catalytic centers, one in each subunit, are represented by the PD. D/EXK motif, in which the two carboxylates are responsible for Mg(2+) binding, the essential cofactor for the great majority of enzymes. The precise mechanism of cleavage has not yet been established for any enzyme, the main uncertainty concerns the number of Mg(2+) ions directly involved in cleavage. Cleavage in the two strands usually occurs in a concerted fashion and leads to inversion of configuration at the phosphorus. The products of the reaction are DNA fragments with a 3'-OH and a 5'-phosphate.

Immigration control of DNA in bacteria: self versus non-self

“…one on each strand of the duplex, is methylated, but modification enzymes function as monomers, an organization consistent with their normal role in the methylation of newly replicated DNA (for reviews see Wilson & Murray, 1991 ;Roberts & Halford, 1993 ;Raleigh & Brooks, 1998 ;Pingoud & Jeltsch, 2001). Genes encoding repressor-like proteins, referred to as C proteins for control, have been identified for some type II R-M systems (Ives et al, 1992 ;Tao et al, 1991 ;Tao & Blumenthal, 1992).…”

How the Bfi I restriction enzyme uses one active site to cut two DNA strands

“…S equence-specific cleavage of double-stranded DNA underpins many genetic events, including recombination, transposition, viral DNA integration, and the restriction of DNA (1)(2)(3)(4). The enzymes that make double-strand breaks use many different strategies to achieve this end.…”

“…The simplest may be the orthodox type II restriction enzymes such as EcoRI or EcoRV, dimers of identical subunits that recognize palindromic DNA sequences. In the presence of Mg 2ϩ , they cleave both strands at fixed positions in their recognition sites (3). They bind their target sites symmetrically, so that one active site from the dimer is positioned against one DNA strand and likewise the second active site on the other strand.…”

Mutational analysis of active‐site residues in the Mycobacterium leprae RecA intein, a LAGLIDADG homing endonuclease: Asp122 and Asp193 are crucial to the double‐stranded DNA cleavage activity whereas Asp218 is not

“…37,40 Several lines of evidence suggest that the conformational changes in the protein and/or DNA often accompany protein-DNA interaction. 41 Fluorescent base analogs, such as 2-AP, are often used to monitor such conformational changes involving proteinnucleic acid interaction. [42][43][44][45] To gain insights into the functional differences in the target DNA selectivity of wild-type PI-MleI and its variants, we used 2-AP fluorescence to monitor changes induced in double-helical DNA by these enzymes.…”