CSHL Poster 4

Structure-Function Differences Among Type I Restriction Endonuclease

DNA Cleavage by EcoR124I is a SAM-Independent Event

Other type I restriction endonucleases require SAM as a cofactor for DNA cleavage. However, R.EcoR124I is unusual in not requiring SAM as a cofactor.

Lane 1, pDRM-1r (single sR124 site) plasmid; Lane 2, pDRM-1R + ENase(R124) + Mg2+; Lane 3, pDRM-1R + ENase(R124) + Mg2+ + SAM; Lane 4, pDRM-1R + ENase(R124) + Mg2+ + ATP; Lane 5, pDRM-1R + ENase(R124) + SAM + ATP; Lane 6, pDRM-1R + ENase(R124) + Mg2++ SAM +ATP; Lane 7, pDRM-1R + ENase(R124) + Mg2+ + ATP + SAH; Lane 8, c.f. lane 7 but higher enzyme conc.; Lane 9, c.f. lane 7 but enzyme 100 x conc.; Lane 10, pDRM-1R + ENase(D50) + Mg2+ +ATP; Lane 11, pDRM-1R + ENase( D50) + Mg2+ + ATP + SAM; Lane 12, pBR322; Lane 13, pBR322 + EcoKI + Mg2+ + ATP; Lane 14, pBR322 + EcoKI + Mg2+ + ATP + SAM.

While EcoKI was found to have an absolute requirement for SAM in order for cleavage of plasmid pBR322 to occur. EcoR124I was found to cleave pDRM-1R in the absence of SAM. This cleavage occurred following long-term storage of EcoR124I.

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