The Fidelity Index provides a systematic quantitation of star activity of DNA restriction endonucleases

Abstract

Restriction endonucleases are the basic tools of molecular biology. Many restriction endonucleases show relaxed sequence recognition, called star activity, as an inherent property under various digestion conditions including the optimal ones. To quantify this property we propose the concept of the Fidelity Index (FI), which is defined as the ratio of the maximum enzyme amount showing no star activity to the minimum amount needed for complete digestion at the cognate recognition site for any particular restriction endonuclease. Fidelity indices for a large number of restriction endonucleases are reported here. The effects of reaction vessel, reaction volume, incubation mode, substrate differences, reaction time, reaction temperature and additional glycerol, DMSO, ethanol and Mn2+ on the FI are also investigated. The FI provides a practical guideline for the use of restriction endonucleases and defines a fundamental property by which restriction endonucleases can be characterized.

Cleavage of Functionalized DNA Containing 5‐Modified Pyrimidines by Type II Restriction Endonucleases

“…In our previous paper [4] we attributed a similar observation of longer products to star activity. [22] We have now found, however, that this observation has a different explanation. Because the reaction mixture still contains some remaining DNA polymerase and dNTPs, the 5'-overhang formed after the cleavage acts as a template for another primer extension reaction that adds several nucleotides to the primary products of cleavage.…”

Diverse Functions of Restriction-Modification Systems in Addition to Cellular Defense

“…Hence, to a large extent, the REases continued to enjoy the status of highly sequence-specific enzymes. However, it is now becoming increas- ingly apparent that REases may not exhibit exquisite sequence specificity after all (141). Instead, many of them may show promiscuous DNA cleavage to various degrees.…”

“…Instead, many of them may show promiscuous DNA cleavage to various degrees. The fidelity index value, which quantitatively measures the star activity of REases, identified a significant number of REases as being "star prone" (141). Additionally, the cleavage of heteroduplex substrates containing mismatches in the target site, when tested with 14 REases, indicated that many enzymes were indeed capable of cleaving mispaired recognition sites (142).…”

Promiscuous restriction is a cellular defense strategy that confers fitness advantage to bacteria

“…Recent studies carried out with >200 REases have revealed that a majority of the REases in the group exhibit relaxed substrate specificity (54,55). Thus, it appears that the catalytic promiscuous activity is an intrinsic property of these enzymes.…”