divalent metal ion differentially regulates the sequential nicking reactions of the giy-yig homing endonuclease i-bmoi二价金属离子不同调节的连续攻击反应giy-yig归巢核酸内切酶i-bmoi.pdfVIP

Divalent Metal Ion Differentially Regulates the Sequential Nicking Reactions of the GIY-YIG Homing Endonuclease I-BmoI 1 ´ ´ 1 1,2 1 Benjamin P. Kleinstiver , Wesley Berube-Janzen , Andrew D. Fernandes , David R. Edgell * 1 Department of Biochemistry, Schulich School of Medicine Dentistry, The University of Western Ontario, London, Ontario, Canada, 2 Department of Applied Mathematics, The University of Western Ontario, London, Ontario, Canada Abstract Homing endonucleases are site-specific DNA endonucleases that function as mobile genetic elements by introducing double-strand breaks or nicks at defined locations. Of the major families of homing endonucleases, the modular GIY-YIG endonucleases are least understood in terms of mechanism. The GIY-YIG homing endonuclease I-BmoI generates a double- strand break by sequential nicking reactions during which the single active site of the GIY-YIG nuclease domain must undergo a substantial reorganization. Here, we show that divalent metal ion plays a significant role in regulating the two independent nicking reactions by I-BmoI. Rate constant determination for each nicking reaction revealed that limiting divalent metal ion has a greater impact on the second strand than the first strand nicking reaction. We also show that substrate mutations within the I-BmoI cleavage site can modulate the first strand nicking reaction over a 314-fold range. Additionally, in-gel DNA footprinting with mutant substrates and modeling of an I-BmoI-substrate complex suggest that amino acid contacts to a critical GC-2 base pair are required to induce a bottom-strand distortion that likely directs conf