Strand-specific contacts and divalent metal ion regulate double-strand break formation by the GIY-YIG homing endonuclease I-BmoI.
GIY-YIG homing endonucleases are modular enzymes consisting of a well-defined N-terminal catalytic domain connected to a variable C-terminal DNA-binding domain. Previous studies have revealed that the role of the DNA-binding domain is to recognize and bind intronless DNA substrate, positioning the N-terminal catalytic domain such that it is poised to generate a staggered double-strand break by an unknown mechanism. Interactions of the N-terminal catalytic domain with intronless substrate are therefore a critical step in the reaction pathway but have been difficult to define. Here, we have taken advantage of the reduced activity of I-BmoI, an isoschizomer of the well-studied bacteriophage T4 homing endonuclease I-TevI, to examine double-strand break formation by I-BmoI. We present evidence demonstrating that I-BmoI generates a double-strand break by two sequential but chemically independent nicking reactions where divalent metal ion is a limiting factor in top-strand nicking. We also show by in-gel footprinting that contacts by the I-BmoI catalytic domain induce significant minor groove DNA distortions that occur independently of bottom-strand nicking. Bottom-strand contacts are critical for accurate top-strand nicking, whereas top-strand contacts have little influence on the accuracy of bottom-strand nicking. We discuss our results in the context of current models of GIY-YIG endonuclease function, with emphasis on the role of divalent metal ion and strand-specific contacts in regulating the activity of a single active site to generate a staggered double-strand break.
Carter
Jonathan M
JM
Department of Biochemistry, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, ON, Canada.
Friedrich
Nancy C
NC
Kleinstiver
Benjamin
B
Edgell
David R
DR
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D003300:Copper / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D004249:DNA Damage; D018983:DNA Footprinting; D024202:Electrophoretic Mobility Shift Assay; D004706:Endodeoxyribonucleases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D007438:Introns / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid
D003300:Copper; D004247:DNA; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease