EcoRII: a restriction enzyme evolving recombination functions?

• Published in: The EMBO journal Vol. 21; no. 19; pp. 5262 - 5268
• Main Authors: Mücke, Merlind, Grelle, Gerlinde, Behlke, Joachim, Kraft, Regine, Krüger, Detlev H., Reuter, Monika
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 01.10.2002; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Base Sequence; Binding Sites; Deoxyribonucleases, Type II Site-Specific - genetics; Deoxyribonucleases, Type II Site-Specific - isolation & purification; Deoxyribonucleases, Type II Site-Specific - metabolism; Deoxyribonucleic acid; DNA; DNA Primers; DNA recombination; DNA restriction; DNA Transposable Elements; EcoRII evolution; Oligodeoxyribonucleotides - metabolism; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Recombination, Genetic; Substrate Specificity; type IIE restriction endonuclease
• ISSN: 0261-4189; 1460-2075
• DOI: 10.1093/emboj/cdf514

The restriction endonuclease EcoRII requires the cooperative interaction with two copies of the sequence 5′CCWGG for DNA cleavage. We found by limited proteolysis that EcoRII has a two‐domain structure that enables this particular mode of protein–DNA interaction. The C‐terminal domain is a new restriction endonuclease, EcoRII‐C. In contrast to the wild‐type enzyme, EcoRII‐C cleaves DNA specifically at single 5′CCWGG sites. Moreover, substrates containing two or more cooperative 5′CCWGG sites are cleaved much more efficiently by EcoRII‐C than by EcoRII. The N‐terminal domain binds DNA specifically and attenuates the activity of EcoRII by making the enzyme dependent on a second 5′CCWGG site. Therefore, we suggest that a precursor EcoRII endonuclease acquired an additional DNA‐binding domain to enable the interaction with two 5′CCWGG sites. The current EcoRII molecule could be an evolutionary intermediate between a site‐specific endonuclease and a protein that functions specifically with two DNA sites such as recombinases and transposases. The combination of these functions may enable EcoRII to accomplish its own propagation similarly to transposons.