Jiao C, Summerlin M, Bruzik KS, Hanakahi L.
Biochemistry. 2015 Sep 23. [Epub ahead of print]
- PMID:
- 26397942
Biochemistry. 2015 Sep 23. [Epub ahead of print]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 participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized.
We report on synthesis, characterization and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at the 2-position of myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by non-homologous 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 (CK2), 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 to investigate the role of IP6 in biological systems. - PMID:
- 26397942
- [PubMed - as supplied by publisher]
2.
Ma Y, Lieber MR.
J Biol Chem. 2002 Mar 29;277(13):10756-9. Epub 2002 Jan 30.
- PMID:
- 11821378
J Biol Chem. 2002 Mar 29;277(13):10756-9. Epub 2002 Jan 30.
Binding of inositol hexakisphosphate (IP6) to Ku but not to DNA-PKcs.Abstract (2002)
The nonhomologous DNA end joining (NHEJ) pathway is responsible for repairing a major fraction of double strand DNA breaks in somatic cells of all multicellular eukaryotes. As an indispensable protein in the NHEJ pathway, Ku has been hypothesized to be the first protein to bind at the DNA ends generated at a double strand break being repaired by this pathway. When bound to a DNA end, Ku improves the affinity of another DNA end-binding protein, DNA-PK(cs), to that end. The Ku.DNA-PK(cs) complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP(6)) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP(6) bound to DNA-PK(cs) (not to Ku). Here we clearly show that, in fact, IP(6) associates not with DNA-PK(cs), but rather with Ku. Furthermore, the binding of DNA ends and IP(6) to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings.
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