Study on the mechanism of the action for type Ⅱ restriction endonucleases

Abstract

Interactions between some TypeII restriction endonucleases and DNA substrates with nicking alterations within or near the recognition sequence of each enzyme were investigated. DNA substrates with a nick at specific positions could be generated by treating the plasmid DNA with endonucleases at lower temperatures(0-4``C). The plasmid DNA, which has a nick within or near the recognition sequence, served as a poor substrate for the endonuclease recognizing the sequence unless the nick was tightly holded by the enzyme: HincII, SalI and SmaI endonucleases cleaved the plasmid DNA substrates specifically nicked by the related isoschizomer, recognizing the same sequence but cleaving different site each other, with much reduced rate compared to the substrates specifically nicked by itself. AvaI could not cleave the plasmid DNA specifically nicked by SmaI at all at the various recommended conditions. Also, nicking alterations in the just outside of the recognition sequence prevent the normal action of the dimeric endonuclease to a considerable extent. In addition, the conditions stabilizing the native conformation of DNA duplex, i.e., low temperature and high ionic strength, made endonucleases act poorly for their cleavage of DNA duplex. The work reported in this thesis the proposed mechanism of the action of TypeII restriction endonucleases with some evidences, particularly in that a conformational shift of duplex DNA within the symmetric DNA-enzyme complex may be required to accomplish the cleavage reaction.