Abstract
The type II restriction endonuclease SsoII shows sequence similarity with 10 other restriction endonucleases, among them the type IIE restriction endonuclease EcoRII, which requires binding to an effector site for efficient DNA cleavage, and the type IIF restriction endonuclease NgoMIV, which is active as a homotetramer and cleaves DNA with two recognition sites in a concerted reaction. We show here that SsoII is an orthodox type II enzyme, which is active as a homodimer and does not require activation by binding to an effector site. Nevertheless, it shares with EcoRII and NgoMIV a very similar DNA-binding site and catalytic center as shown here by a mutational analysis, indicative of an evolutionary relationship between these three enzymes. We suggest that a similar relationship exists between other orthodox type II, type IIE, and type IIF restriction endonucleases. This may explain why similarities may be more pronounced between members of different subtypes of restriction enzymes than among the members of a given subtype.
Highlights
More than 3000 different type II restriction endonucleases are known and characterized with respect to their cleavage specificities [1]
Our interest in studying this enzyme is due to its sequence similarity to EcoRII, a type IIE enzyme, which being somewhat larger than SsoII is a dimer of identical subunits each consisting of 404 amino acid residues [20, 21]
As a type IIE enzyme EcoRII has two DNA-binding sites, one associated with the catalytic center and the other serving as an effector site [22] [23,24,25], similar to that shown for NaeI (26 –29)
Summary
More than 3000 different type II restriction endonucleases are known and characterized with respect to their cleavage specificities [1]. Based on sequence alignments and structural comparisons and verified by a mutational analysis, we have identified two regions that are essential for the function of SsoII, one of which is very likely to harbor the catalytic center The sequences characterizing these regions are present in several other orthodox type II as well as type IIE enzymes (which require binding of a substrate at the active site and the effector site) and IIF enzymes (which require binding of a substrate at two active sites formed by four identical subunits) that recognize CCGG, CCNGG, or CCWGG in different sequence contexts, an observation that has implications for the evolution of these subtypes of type II restriction endonucleases
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