title: Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.
abstract: Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems.
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago , 833 South Wood Street (M/C 781), Chicago, Illinois 60612, United States.
authors.forename: Chensong
authors.initials: C
authors.lastname: Jiao
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago , Rockford Health Sciences Campus, 1601 Parkview Avenue, Rockford, Illinois 61107, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Summerlin
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago , 833 South Wood Street (M/C 781), Chicago, Illinois 60612, United States.
authors.forename: Karol S
authors.initials: KS
authors.lastname: Bruzik
authors.affiliation: Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois, Chicago , Rockford Health Sciences Campus, 1601 Parkview Avenue, Rockford, Illinois 61107, United States.
authors.forename: Leslyn
authors.initials: L
authors.lastname: Hanakahi
mesh_terms: D019921:Biotinylation; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D004265:DNA Helicases / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D000072200:Ku Autoantigen; D010833:Phytic Acid / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D010833:Phytic Acid; D004265:DNA Helicases; C106893:XRCC5 protein, human; D000072200:Ku Autoantigen