Processive cleavage of concatemer DNA duplexes by Eco RII restriction endonuclease

Abstract

Eco RII restriction endonuclease cleaves synthetic DNA-duplexes in which the recognition sites of this enzyme (5'...CCATGG...) are repeated every 9 base pairs with the alternating orientation of the central AT pair. It operates in a processive mode, i.e. the bound enzyme molecule slides along the substrate toward neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained neighboring recognition sites. Nona-nucleotides are the main products of the cleavage. The data obtained point to the capability of Eco RII endonuclease to recognize and cleave the substrate under both possible orientations of the central AT-pair of the recognition site with respect to the bound enzyme molecule. These data also show the close similarity of DNA structures in a complex with the enzyme and without.

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages

“…These findings allow one to suppose that the hydrolysis of two DNA strands by endonuclease EcoRII is catalysed by two identical hydrolytical active centers operating independently. It is consistent with the 'symmetric model" of the enzyme-substrate complex [16,17] taken into consideration for EcoRII endonuclease [14]. This model implies, in particular, binding of the dimeric restriction enzyme to both strands of DNA recognition site and a symmetrical arrangement of two hydrolytical active centers near two scissile bonds.…”

“…Very slow cleavage of DNA duplex XII enables us to conclude that rather extended DNA frag-ment is necessary for normal cleavage of the substrate. According to our earlier estimation [14], EcoRII endonuclease binds to the DNA fragment of 23-32 base pairs long.…”

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. VI. The binding and cleavage of substrates containing nucleotide analogs

“…This fact indicates that there is no preferential cleavage of unmodified or modified strand for polymers II and III. In the case of polymer I (see also our previous paper [15]) and polymer III nonanucleotides prevail even at a low degree of hydrolysis. For polymer II the nonanucleotide content is appreciably lower.…”

“…Then the gel was autoradiographed and the radioactivity of the gel slices that corresponded to the intact substrate and the reaction productsnonanucleotides and their oligomers (k-mers) was determined by Cherenkov counting. These data were used for computing the relative mass content of each of the k-mers (Mk) and the amount of cleaved bonds as described previously [15].…”

“…As seenin Fig. 2, polymers II and III, like polymer I [8,15], are cleaved by EcoRII enzyme and yield nonanucleotides and 18, 27, 35, etc.-membered products. Polymer IV is not cleaved detectably under the same conditions.…”