Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6‑Binding Proteins

• Published in: Biochemistry (Easton) 2015-10, Vol.54 (41), p.6312-6322
• Main Authors: Jiao, Chensong, Summerlin, Matthew, Bruzik, Karol S, Hanakahi, Leslyn
• Format: Article
• Language: English
• Subjects: Biotinylation; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Helicases - metabolism; HEK293 Cells; HeLa Cells; Humans; Ku Autoantigen; Phytic Acid - chemical synthesis; Phytic Acid - chemistry; Phytic Acid - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/acs.biochem.5b00642

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.