Binding of Inositol Hexakisphosphate (IP6) to Ku but Not to DNA-PKcs

Yunmei Ma,Michael R Lieber

doi:10.1074/jbc.c200030200

Yunmei Ma, Michael R Lieber

Open Access

https://doi.org/10.1074/jbc.c200030200

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Abstract

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.

Highlights

From the Norris Comprehensive Cancer Center, Departments of Pathology, Biochemistry & Molecular Biology, Molecular Microbiology & Immunology, and Biological Sciences, University of Southern California Keck School of Medicine, Los Angeles, California 90089-9176
IP6 showed no association with DNA-PKcs alone, above the low level of background binding to the protein G-Sepharose beads (Fig. 1A, histogram bar 2 versus bar 1)
Ku Binds to IP6—These results demonstrate that IP6, but not IP3, binds to Ku, and neither IP6 nor IP3 associate with DNA-PKcs

Summary

Accelerated Publication

The second nuclear role of inositol phosphates was in NHEJ in nuclear extracts in which compatible DNA ends were studied for joining [25] In this latter study, it was reported that inositol hexakisphosphate stimulates the joining of complementary DNA ends in a cell-free system and associates with DNA-PK based on the co-elution of IP6 and DNA-PK kinase activity from a gel filtration column. It was reported that inositol hexakisphosphate stimulates the joining of complementary DNA ends in a cell-free system and associates with DNA-PK based on the co-elution of IP6 and DNA-PK kinase activity from a gel filtration column It was not determined in this study whether IP6 was binding to the 469kDa DNA-PKcs or to the Ku that was in the DNA-PK preparation. The IP6 binds to Ku but not to DNA-PKcs, and the binding of DNA ends and IP6 to Ku seem to be independent of each other

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION