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

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-PKcs, to that end. The Ku·DNA-PKcs complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP6) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP6 bound to DNA-PKcs (not to Ku). Here we clearly show that, in fact, IP6 associates not with DNA-PKcs, but rather with Ku. Furthermore, the binding of DNA ends and IP6 to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings. 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-PKcs, to that end. The Ku·DNA-PKcs complex is often termed the DNA-PK holoenzyme. It was recently shown that myo-inositol hexakisphosphate (IP6) stimulates the joining of complementary DNA ends in a cell free system. Moreover, the binding data suggested that IP6 bound to DNA-PKcs (not to Ku). Here we clearly show that, in fact, IP6 associates not with DNA-PKcs, but rather with Ku. Furthermore, the binding of DNA ends and IP6 to Ku are independent of each other. The possible relationship between inositol phosphate metabolism and DNA repair is discussed in light of these findings. Double strand DNA breaks are among the most lethal DNA lesions, and they arise in somatic cells of multicellular eukaryotes spontaneously in the absence of external factors (1.Karanjawala Z.E. Grawunder U. Hsieh C.-L. Lieber M.R. Curr. Biol. 1999; 9: 1501-1504Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 2.vanGent D.C. Hoeijmakers J.H.J. Kanaar R. Nat. Rev. Genet. 2001; 2: 196-206Crossref PubMed Scopus (962) Google Scholar). Recently, we demonstrated that oxidative metabolism is the cause of at least a substantial fraction of these breaks (3.Karanjawala Z. Murphy N. Hinton D.R. Hsieh C.-L. Lieber M.R. Curr. Biol. 2002; 12: 397-402Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar). There are two pathways for repairing chromosome breaks (4.Kanaar R. Hoeijmakers J.H.J. Genes Function. 1997; 1: 165-174Crossref PubMed Scopus (50) Google Scholar). Homologous recombination repairs breaks that arise during late S and G2 of the cell cycle in vertebrate cells (5.Takata M. Sasaki M.S. Sonoda E. Morrison C. Hashimoto M. Utsumi H. Yamaguchi-Iwai Y. Shinohara A. Takeda S. EMBO J. 1998; 17: 5497-5508Crossref PubMed Scopus (1008) Google Scholar). Nonhomologous DNA end joining (NHEJ) 1The abbreviations used are: NHEJnonhomologous DNA end joiningDNA-PKDNA-dependent protein kinaseIP6myo-inositol hexakisphosphateIP3myo-inositol 1,4,5-trisphosphateBSAbovine serum albumindsdouble strandEMSAelectrophoretic mobility shift assaySPRsurface plasmon resonance repairs double strand DNA breaks that arise during G0, G1, and early S phases of the vertebrate cell cycle (5.Takata M. Sasaki M.S. Sonoda E. Morrison C. Hashimoto M. Utsumi H. Yamaguchi-Iwai Y. Shinohara A. Takeda S. EMBO J. 1998; 17: 5497-5508Crossref PubMed Scopus (1008) Google Scholar). nonhomologous DNA end joining DNA-dependent protein kinase myo-inositol hexakisphosphate myo-inositol 1,4,5-trisphosphate bovine serum albumin double strand electrophoretic mobility shift assay surface plasmon resonance The NHEJ repair process is thought to begin with the binding of a protein called Ku, which consists of Ku70 (70 kDa) and Ku86 (83 kDa) subunits, to DNA ends (6.Lieber M.R. Genes Cells. 1999; 4: 77-85Crossref PubMed Scopus (147) Google Scholar). Ku improves the DNA end binding affinity of the 469-kDa DNA-dependent protein kinase (DNA-PKcs) (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). DNA-PKcs is a serine/threonine protein kinase that is only active when bound to DNA ends (8.Anderson C.W. Carter T.H. Jessberger R. Lieber M.R. Molecular Analysis of DNA Rearrangements in the Immune System. Springer-Verlag, Heidelberg, Germany1996: 91-112Google Scholar). When Ku and DNA-PKcs are both bound at the same DNA end, the complex is referred to as the DNA-PK holoenzyme, whereas the 469-kDa DNA-PKcs is termed the catalytic subunit (hence the subscript designation, cs) (9.Gottlieb T. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar). The physiologic phosphorylation target of the 469-kDa DNA-PKcs has not been identified (10.Anderson C.W. Lees-Miller S.P. Crit. Rev. Eukaryot. Gene Expr. 1992; 2: 283-314PubMed Google Scholar). After the DNA ends are trimmed into a form that is ligatable, a complex of XRCC4 and DNA ligase IV is responsible for carrying out the ligation specifically of double-strand breaks (11.Wilson T.E. Grawunder U. Lieber M.R. Nature. 1997; 388: 495-498Crossref PubMed Scopus (345) Google Scholar, 12.Grawunder U. Lieber M.R. Nucleic Acids Res. 1997; 25: 1375-1382Crossref PubMed Scopus (37) Google Scholar, 13.Gao Y. Sun Y. Frank K. Dikkes P. Fujiwara Y. Seidl K. Sekiguchi J. Rathbun G. Swat W. Wang J. Bronson R. Malynn B. Bryans M. Zhu C. Chaudhuri J. Davidson L. Ferrini R. Stamato T. Orkin S.H. Greenberg M.E. Alt F.W. Cell. 1998; 95: 891-902Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar, 14.Grawunder U. Zimmer D. Fugmann S. Schwarz K. Lieber M.R. Mol. Cell. 1998; 2: 477-484Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar, 15.Barnes D.E. Stamp G. Rosewell I. Denzel A. Lindahl T. Curr. Biol. 1998; 8: 1395-1398Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar, 16.Schar P. Herrmann G. Daly G. Lindahl T. Genes Devel. 1997; 11: 1912-1924Crossref PubMed Scopus (176) Google Scholar, 17.Teo S.H. Jackson S.P. EMBO J. 1997; 16: 4788-4795Crossref PubMed Scopus (232) Google Scholar). Evidence for the importance of Ku in eukaryotic NHEJ has been documented by a large body of genetic evidence (reviewed in Refs. 6.Lieber M.R. Genes Cells. 1999; 4: 77-85Crossref PubMed Scopus (147) Google Scholar,18.Lieber M.R. Am. J. Pathol. 1998; 153: 1323-1332Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, and 19.Featherstone C. Jackson S.P. Mutat. Res. 1999; 434: 3-15Crossref PubMed Scopus (241) Google Scholar). Cells genetically deficient for either subunit of Ku are ionizing radiation sensitive. In addition, they are unable to carry out the rejoining phase of V(D)J recombination, which is a physiologic DNA double strand breakage and rejoining process restricted to lymphoid cells. The same phenotypic characteristics apply to other NHEJ components. In vitro, Ku has a high affinity for DNA ends (KD = 10−9) (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar, 20.Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar) and binds other single to double strand DNA transitions (21.Smider V. Rathmell W.K. Brown G. Lewis S. Chu G. Mol. Cell. Biol. 1998; 18: 6853-6858Crossref PubMed Google Scholar). Inositol phosphate metabolism is a known form of intracellular signaling, primarily in the cytoplasm (22.Irvine R.F. Schell M.J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 327-338Crossref PubMed Scopus (541) Google Scholar). There are only two lines of work describing effects of inositol phosphates on nuclear processes. One set of studies describes the roles of inositol phosphates on mRNA transport and transcriptional regulation (23.York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 24.Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). The second nuclear role of inositol phosphates was in NHEJ in nuclear extracts in which compatible DNA ends were studied for joining (25.Hanakahi L. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). 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 not determined in this study whether IP6 was binding to the 469-kDa DNA-PKcs or to the Ku that was in the DNA-PK preparation. However, the homology of DNA-PKcs to proteins related to phosphatidylinositol metabolism was taken as an implicit indication that the IP6 binds to the DNA-PKcs rather than to Ku. Here we demonstrate that, in fact, 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. IP6 and myo-inositol 1,4,5-trisphosphate (IP3) were purchased from Sigma. Tritiated IP6([3H]IP6, 20.5 Ci/mmol) and IP3([3H]IP3, 21.0 Ci/mmol) were obtained from PerkinElmer Life Sciences. Native DNA-PKcs was purified as described previously (26.Chan D.W. Mody C.H. Ting N.S. Lees-Miller S.P. Biochem. Cell Biol. 1996; 74: 67-73Crossref PubMed Scopus (83) Google Scholar) except that HeLa cells were used as the source for purification. C-terminal His-tagged Ku70 and nontagged Ku86 were co-expressed in the baculovirus system and purified as described previously (27.Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (269) Google Scholar). Nontagged Ku was expressed and purified as described previously (28.Walker J.R. Corpina R.A. Goldberg J. Nature. 2001; 412: 607-614Crossref PubMed Scopus (878) Google Scholar) (a gift of Dr. J. Goldberg). The concentration of purified proteins was estimated by comparing to bovine serum albumin (BSA) standards on a Coomassie Blue stained SDS-PAGE gel. Immuno pull-down of IP6 was performed in 50-μl reactions that contain 10 mm Tris, pH 7.5, 50 mm NaCl, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol, 10% glycerol, and 0.1 mg/ml BSA. Anti-Ku70 monoclonal antibody (clone N3H10, NeoMarkers, Fremont, CA), anti-Ku86 monoclonal antibody (clone 111, NeoMarkers), and anti-DNA-PKcs monoclonal antibodies (clone 42-27 and 25-4) were used as indicated to reach a final total antibody amount of 20 μg in each reaction. The rest of the reagents were added to each 50-μl reaction as indicated: 1 pmol of [3H]IP6 or [3H]IP3, 25–50 pmol of 35-bp DNA YM-8/YM-9 (blunt ds DNA, YM-8 5′-AGG CTG TGT TAA GTA TCT GCG CTC GCC CTC AGA GG-3′), 6.2–10 pmol of Ku, and 2.5 pmol of DNA-PKcs. Then, 100 μl of 50% slurry of protein G-Sepharose (Amersham Biosciences) and 350 μl of binding buffer were added to make the final volume 500 μl. The pull-down reactions were allowed to proceed for 1 h at 4 °C with constant mixing. After washing in the same binding buffer (without BSA) for 4 times (0.8–1 ml of buffer for each time), the beads were resuspended in 200 μl (2 × 100 μl) of binding buffer (without BSA) then mixed into 2 ml of ScintiVerse II scintillation fluid (Fisher Scientific). The amounts of bead-associated 3H were measured in a liquid scintillation analyzer (model Tri-Carb 2100TR, Packard BioScience, Meriden, CT). In each 50-μl reaction (contains the same buffer components as the immuno pull-down reactions), 50 pmol of 5′-biotinylated 35-bp DNA YM-8/YM-9, 6.2 pmol of Ku, and 1 pmol of inositol phosphate were used. 100 μl of 50% streptavidin agarose (Sigma) were then added into each reaction. The binding, washing, and scintillation counting steps were carried out as described above. The gel shift assay was performed as described previously (20.Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar), and 0.5 nm labeled YM-6/YM-7 (20.Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar) and 1 nm Ku were used for all the reactions. IP6 and IP3 were diluted in H2O first and then added to the reaction mixtures to the indicated final concentrations. The SPR experiment was done as described previously (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar, 20.Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar). A 5′-biotinylated 35-bp DNA (sequence is the same as YM-8/YM-9) was immobilized on the streptavidin-coated surface of the sensor chip (Sensor chip SA, Biacore, San Diego, CA). Ku was diluted in the running buffer and injected at a flow rate of 5 μl/min for 4 min. The injection of Ku was repeated to ensure that the surface-immobilized DNA on the sensor chip was saturated with Ku. Then DNA-PKcs (to a final concentration of 6.1 nm) and additional reagents (1 mm ATP, 1 mmMgCl2, and 0.1 mm IP6 or IP3, final concentration indicated) were mixed in the running buffer and injected at 5 μl/min for 4 min. Proteins bound to the sensor chip were allowed to dissociate for 6 min before the surface was regenerated with 0.05% SDS. The resulting sensorgrams were edited using the BIAevaluation software (version 3.0). A previous study on the association of DNA-PK and IP6 used a DNA-PK preparation that contained Ku and, possibly, contaminating DNA fragments (25.Hanakahi L. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). We were interested in testing whether binding of IP6 to DNA-PK was dependent on Ku and/or DNA ends. Native DNA-PKcs and recombinant Ku were purified as described previously. DNA-PKcs was immobilized on protein G-Sepharose beads via monoclonal antibodies against it. Tritiated-IP6was incubated with these Immunobeads in the absence or presence of Ku and a 35-bp DNA (under the buffer conditions specified under “Experimental Procedures”). The DNA length of 35 bp was chosen, because this permits the binding of Ku and DNA-PKcssimultaneously to the same DNA molecule (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). The radioactivity associated with the extensively washed beads was then measured using a liquid scintillation counter. Surprisingly, 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). This lack of association between IP6 and DNA-PKcs was obtained regardless of the presence of the 35-bp linear DNA (Fig. 1A, bar 3 versus bar 2). When IP6 was added to DNA-PKcs immunobeads along with Ku, there was only a near background level of binding detcted (Fig. 1A, bar 4), in agreement with our observation that Ku does not associate with DNA-PKcs in the absence of DNA (see below). Interestingly, when the binding of IP6 to DNA-PKcs was tested in the presence of Ku and 35-bp DNA, a substantial amount of binding was observed (Fig. 1A, bar 5). This could be because Ku induces a conformational change in DNA-PKcs to permit its binding of IP6. Alternatively, IP6 might bind at the interface between Ku and DNA-PKcs when both are present on DNA, because 35 bp is long enough for Ku and DNA-PKcs to co-localize (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). Alternatively, IP6 may simply bind to Ku. To test this, we omitted DNA-PKcs entirely and examined the binding of IP6 to bead-immobilized Ku via monoclonal anti-Ku antibodies. In this case, we observed a very high level of IP6 binding (Fig. 1A, bar 6). The specificity of binding of IP6 to Ku was tested with another inositol IP3, and only a background level of binding was (Fig. 1A, bar it that IP6 was in binding to Ku rather than to DNA-PKcs We to out the that the affinity on Ku70 might be responsible for the association between IP6 and recombinant Ku. To test this, we the binding of IP6 to purified Ku with and the and the association of IP6 to these of Ku was (Fig. the binding of IP6 to Ku is by the affinity To the role of DNA in the between IP6 and Ku, we used a monoclonal antibody against Ku70 to the Ku The level of binding was regardless of whether the 35-bp ds DNA was present (Fig. 1 and 2). with an anti-Ku86 monoclonal antibody (Fig. 3 and As shown the binding of IP6 to Ku was because IP3 to bind (Fig. bar 5). As was the for anti-DNA-PKcs monoclonal antibodies (Fig. and of the anti-Ku antibodies bound to IP6 not to IP3 (Fig. bar 5). data clearly that IP6 binds to Ku rather than to DNA-PKcs, and this binding is independent of the presence of evidence for the binding of IP6 to Ku be observed when the are in a a linear DNA was immobilized on beads via a and Ku was added to these DNA with the IP3 not (Fig. 1 and 2). The beads with DNA could the complex as as protein G-Sepharose with anti-Ku antibodies (Fig. bar 1 versus 3 and 5). This is additional evidence that IP6 binds to Ku, and this is by We and previously done studies of the binding of Ku to DNA ends. Recently, we showed that on linear DNA long enough to bind two Ku linear the second Ku molecule with a constant (20.Ma Y. Lieber M.R. Biochemistry. 2001; 40: 9638-9646Crossref PubMed Scopus (24) Google Scholar). the two Ku bind to a DNA We were interested in testing whether the binding of the complex this DNA be with the of Ku the same To test this, of Ku and DNA were that 50% of Ku bind to under these each DNA molecule either or two Ku The concentration of IP6 was then a that the physiologic concentration of IP6 in eukaryotic cells. the first Ku binding the second Ku binding was by IP6 (Fig. it does not that IP6 the association of Ku with DNA ends. Ku has been of DNA-PK to the that it the binding of DNA-PKcs to a DNA end and stimulates the kinase activity of DNA-PKcs (9.Gottlieb T. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar). the binding of IP6 to Ku might the fraction of is in a complex with Ku and The kinase activity of DNA-PK on a is not by IP6 (25.Hanakahi L. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). To the that IP6 might or the binding of DNA-PKcs to a DNA end, an SPR experiment was The SPR the of the in the of associated with a In our a 35-bp ds DNA was immobilized on a streptavidin-coated Ku was then allowed to bind to the free end of the DNA on the surface to or near by two of the same Ku The binding of DNA-PKcs was tested in the presence of IP6 or the association phase phase with the injection of the phase phase with the of was by IP6. This that IP6 does not DNA-PKcs in the association with the demonstrate that but not IP3, binds to Ku, and IP6 IP3 associate with DNA-PKcs. work indicated that the of DNA end joining in a cell system that was to of DNA-PKcs (25.Hanakahi L. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar). In that study when the of IP6 and DNA-PK was by a gel filtration of IP6 by scintillation counting of each and the of DNA-PK by DNA-PK activity of each In light of our which that Ku associates with for the observation could be that there was contaminating DNA in the DNA-PK preparation that allowed the association of Ku and the association of IP6 and We previously shown that DNA-PKcs and Ku not associate in the absence of DNA ends (27.Yaneva M. Kowalewski T. Lieber M.R. EMBO J. 1997; 16: 5098-5112Crossref PubMed Scopus (269) Google Scholar), and this observation has been by our (7.West R.B. Yaneva M. Lieber M.R. Mol. Cell. Biol. 1998; 18: 5908-5920Crossref PubMed Scopus (145) Google Scholar). In the study, the to the complex by DNA-PKcs immunobeads (Fig. bar this of by may be to low of contaminating DNA that with DNA-PKcs, steps are not to the The contaminating DNA then to bind both Ku and DNA-PKcs on the same that bind DNA-PKcs than DNA bind to DNA-PKcs. IP6 was reported to the of joining of compatible DNA ends by cell extracts (25.Hanakahi L. Bartlet-Jones M. Chappell C. Pappin D. West S.C. Cell. 2000; 102: 721-729Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar), and our study that this might association with Ku. One possible of this could be that IP6 of Ku. A second is that the complex might with other factors in a that the NHEJ could be Ku has been reported to and with DNA-PKcs in presence of DNA (9.Gottlieb T. Jackson S.P. Cell. 1993; 72: 131-142Abstract Full Text PDF PubMed Scopus (1027) Google Scholar), DNA ligase complex J. Mol. Cell. Biol. 2000; PubMed Scopus Google Scholar, L. K. P. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), and the A. D. Genes 2000; Google Scholar, B. L. J. Biol. 2000; Full Text Full Text PDF PubMed Scopus (176) Google Scholar). The of the of of these might in a on the of The of the of We Dr. S. C. and of the Lieber the early phases of this in Dr. R. Lieber and Dr. West as it that both that IP6 and Ku