Sequential Loading of Saccharomyces cerevisiae Ku and Cdc13p to Telomeres
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m809131200
ISSN1083-351X
AutoresTzung-Ju Wu, Yi-Hsuan Chiang, Yi-Chien Lin, Chang-Ru Tsai, Tai-Yuan Yu, Ming-Ta Sung, Yan‐Hwa Wu Lee, Jing‐Jer Lin,
Tópico(s)CRISPR and Genetic Engineering
ResumoKu is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C1–3(A/T)G1–3 telomeric DNA repeats plus single-stranded TG1–3 telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres. Ku is a heterodimeric protein involved in nonhomologous end-joining of the DNA double-stranded break repair pathway. It binds to the double-stranded DNA ends and then activates a series of repair enzymes that join the broken DNA. In addition to its function in DNA repair, the yeast Saccharomyces cerevisiae Ku (Yku) is also a component of telomere protein-DNA complexes that affect telomere function. The yeast telomeres are composed of duplex C1–3(A/T)G1–3 telomeric DNA repeats plus single-stranded TG1–3 telomeric DNA tails. Here we show that Yku is capable of binding to a tailed-duplex DNA formed by telomeric DNA that mimics the structure of telomeres. Addition of Cdc13p, a single-stranded telomeric DNA-binding protein, to the Yku-DNA complex enables the formation of a ternary complex with Cdc13p binding to the single-stranded tail of the DNA substrate. Because pre-loading of Cdc13p to the single-stranded telomeric tail inhibits the binding of Yku, the results suggested that loading of Yku and Cdc13p to telomeres is sequential. Through generating a double-stranded break near telomeric DNA sequences, we found that Ku protein appears to bind to the de novo synthesized telomeres earlier than that of Cdc13p in vivo. Thus, our results indicated that Yku interacts directly with telomeres and that sequential loading of Yku followed by Cdc13p to telomeres is required for both proteins to form a ternary complex on telomeres. Our results also offer a mechanism that the binding of Cdc13p to telomeres might prevent Yku from initiating DNA double-stranded break repair pathway on telomeres. DNA damages in the form of double-stranded breaks (DSBs) 4The abbreviations used are: DSB, double-stranded break; EMSA, electrophoretic mobility shift assay; Ni-NTA, nickel-nitrilotriacetic acid; r, recombinant. 4The abbreviations used are: DSB, double-stranded break; EMSA, electrophoretic mobility shift assay; Ni-NTA, nickel-nitrilotriacetic acid; r, recombinant. compromise the integrity of genomes. Failure in repairing or mis-repairing double-stranded breaks can lead to chromosome instability and eventually cell death or cancer (1O'Driscoll M. Jeggo P.A. Nat. Rev. Genet. 2006; 7: 45-54Crossref PubMed Scopus (461) Google Scholar). Double-stranded breaks are repaired by two main pathways, the homologous recombination and nonhomologous DNA end-joining. In nonhomologous DNA end-joining, Ku is the first protein to bind to the DNA ends to initiate the repair pathway (2Lieber M.R. Ma Y. Pannicke U. Schwarz K. Nat. Rev. Mol. Cell Biol. 2003; 4: 712-720Crossref PubMed Scopus (788) Google Scholar). Upon binding, Ku then recruits a series of repair enzymes to join the broken ends (2Lieber M.R. Ma Y. Pannicke U. Schwarz K. Nat. Rev. Mol. Cell Biol. 2003; 4: 712-720Crossref PubMed Scopus (788) Google Scholar). Ku is a heterodimeric protein composed of 70- and ∼80-kDa subunits. In Saccharomyces cerevisiae, Ku includes Yku70 and Yku80 subunits. Because the biochemical configuration of the broken ends could be very diverse on DSBs, Ku binds to double-stranded ends in a sequence- and energy-independent manner. It is capable of binding to DNA ends with blunt 3′-overhangs or 5′-overhangs as well as double-stranded DNA with nicks, gaps, or internal loops (3Mimori T. Hardin J.A. J. Biol. Chem. 1986; 261: 10375-10379Abstract Full Text PDF PubMed Google Scholar, 4Paillard S. Strauss F. Nucleic Acids Res. 1991; 19: 5619-5624Crossref PubMed Scopus (188) Google Scholar, 5Falzon M. Fewell J.W. Kuff E.L. J. Biol. Chem. 1993; 268: 10546-10552Abstract Full Text PDF PubMed Google Scholar, 6Ono M. Tucker P.W. Capra J.D. Nucleic Acids Res. 1994; 22: 3918-3924Crossref PubMed Scopus (72) Google Scholar, 7Walker J.R. Corpina R.A. Goldberg J. Nature. 2001; 412: 607-614Crossref PubMed Scopus (869) Google Scholar). However, Ku does not have high affinity to single-stranded DNA. The crystal structure of human Ku heterodimer indicates that it forms a ring structure that encircles duplex DNA (7Walker J.R. Corpina R.A. Goldberg J. Nature. 2001; 412: 607-614Crossref PubMed Scopus (869) Google Scholar). This unique structure feature enables Ku to recognize DNA ends and achieves its high affinity binding. In additional to the role in double-stranded break repair, Ku was shown to be a component of telomeric protein-DNA complex in yeast and mammals (8Hsu H.-L. Gilley D. Blackburn E.H. Chen D.J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12454-12458Crossref PubMed Scopus (274) Google Scholar, 9Gravel S. Larrivee M. Labrecque P. Wellinger R.J. Science. 1998; 280: 741-744Crossref PubMed Scopus (365) Google Scholar, 10d'Adda di Fagagna F. Hande M.P. Tong W.M. Roth D. Lansdorp P.M. Wang Z.Q. Jackson S.P. Curr. Biol. 2001; 11: 1192-1196Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar). Telomeres are terminal structures of chromosomes composed of short tandem repeated sequences (11Blackburn E.H. Greider C.W. Szostak J.W. Nat. Med. 2006; 12: 1133-1138Crossref PubMed Scopus (679) Google Scholar, 12Blackburn E.H. J. Biol. Chem. 1990; 265: 5919-5921Abstract Full Text PDF PubMed Google Scholar). Mutation of YKU70 or YKU80 causes defects in telomere structure (13Boulton S.J. Jackson S.P. Nucleic Acids Res. 1996; 24: 4639-4648Crossref PubMed Scopus (410) Google Scholar, 14Porter S.E. Greenwell P.W. Ritchie K.B. Petes T.D. Nucleic Acids Res. 1996; 24: 582-585Crossref PubMed Scopus (202) Google Scholar, 15Bianchi A. de Lange T. J. Biol. Chem. 1999; 274: 21223-21227Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar), telomere silencing (16Nugent C.I. Bosco G. Ross L.O. Evans S.K. Salinger A.P. Moore J.K. Haber J.E. Lundblad V. Curr. Biol. 1998; 8: 657-660Abstract Full Text Full Text PDF PubMed Google Scholar, 17Laroche T. Martin S.G. Gotta M. Gorham H.C. Pryde F.E. Louis E.J. Gasser S.M. Curr. Biol. 1998; 8: 653-656Abstract Full Text Full Text PDF PubMed Google Scholar, 18Boulton S.J. Jackson S.P. EMBO J. 1998; 17: 1819-1828Crossref PubMed Scopus (554) Google Scholar, 19Mishra K. Shore D. Curr. Biol. 1999; 9: 1123-1126Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar), and replication timing of telomeres (20Cosgrove A.J. Nieduszynski C.A. Donaldson A.D. Genes Dev. 2002; 16: 2485-2490Crossref PubMed Scopus (83) Google Scholar). The function of yeast Ku (Yku) on telomeres could mediate through protein-protein interaction with Sir4p or protein-RNA interaction with Tlc1 RNA (21Tsukamoto Y. Kato J. Ikeda H. Nature. 1997; 388: 900-903Crossref PubMed Scopus (310) Google Scholar, 22Stellwagen A.E. Haimberger Z.W. Veatch J.R. Gottschling D.E. Genes Dev. 2003; 17: 1-12Crossref PubMed Scopus (236) Google Scholar). For example, through the interaction with Sir4p, Yku selectively affects telomeres silencing but not the silent mating type loci (17Laroche T. Martin S.G. Gotta M. Gorham H.C. Pryde F.E. Louis E.J. Gasser S.M. Curr. Biol. 1998; 8: 653-656Abstract Full Text Full Text PDF PubMed Google Scholar). Yku could also bind to telomerase Tlc1 RNA for telomere length maintenance (22Stellwagen A.E. Haimberger Z.W. Veatch J.R. Gottschling D.E. Genes Dev. 2003; 17: 1-12Crossref PubMed Scopus (236) Google Scholar). Judged by the DNA binding activity of Yku, it is reasonable to suggest that it may bind directly to telomeric DNA. Indeed, it was shown that human Ku is capable of binding directly to telomeric DNA in vitro (15Bianchi A. de Lange T. J. Biol. Chem. 1999; 274: 21223-21227Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Moreover, because the deletion of SIR4 in budding yeast (23Martin S.G. Laroche T. Suka N. Grunstein M. Gasser S.M. Cell. 1999; 97: 621-633Abstract Full Text Full Text PDF PubMed Scopus (402) Google Scholar) or Taz1 in fission yeast (24Miyoshi T. Sadaie M. Kanoh J. Ishikawa F. J. Biol. Chem. 2003; 278: 1924-1931Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) does not abolish the association of Ku with chromosomal ends, this suggests that Ku might bind directly to telomeric DNA in cells. However, because yeast telomeres have a short 12–14-mer single-stranded tail (25Larrivee M. LeBel C. Wellinger R.J. Genes Dev. 2004; 18: 1391-1396Crossref PubMed Scopus (189) Google Scholar), it is uncertain whether Yku could pass the single-stranded region to reach its binding site. The direct binding of Yku to telomeric DNA has not been experimentally determined. In contrast to double-stranded breaks, the ends of linear chromosomes are not recognized by repair enzymes as DNA damage. In S. cerevisiae, Cdc13p is the single-stranded TG1–3 DNA-binding protein that enables cells to differentiate whether the ends of a linear DNA are telomeres or broken ends (26Lin J.-J. Zakian V.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13760-13765Crossref PubMed Scopus (273) Google Scholar, 27Nugent C.I. Hughes T.R. Lue N.F. Lundblad V. Science. 1996; 274: 249-252Crossref PubMed Scopus (513) Google Scholar, 28Bourns B.D. Alexander M.K. Smith A.M. Zakian V.A. Mol. Cell. Biol. 1998; 18: 5600-5608Crossref PubMed Scopus (116) Google Scholar, 29Pang T.-L. Wang C.Y. Hsu C.-L. Chen M.Y. Lin J.-J. J. Biol. Chem. 2003; 278: 9318-9321Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Thus, although the mechanism of how cells prevent the activation of DSB repair pathway in telomere is unclear, it is likely that binding of Cdc13p to telomeres might inhibit the initiation of DNA damage response by the Ku protein. Here, using a tailed-duplex DNA synthesized by telomeric DNA sequences to mimic telomere structure, we showed that Yku binds directly to this tailed-duplex DNA substrate and forms a ternary complex with Cdc13p. Our results also showed that Yku loaded to a de novo synthesized telomere earlier than Cdc13p in vivo. These results support the direct binding of Yku to telomeric DNA and that the spatial orientation of Cdc13p might block the activation of DSB repair pathway on telomeres. Purification of Yku from Yeast—Yeast BJ2168 cells (MATa leu2 trp1 ura3-52 prb1-1122 pep4-3 prc1-407 gal2) carrying pRS425TEF-YKU70-TAP and pRS423TEF-YKU80-TAP was employed to isolate Yku heterodimer from yeast (A gift from D. E. Gottschling, Fred Hutchinson Cancer Research Center, Seattle). Purification of the Yku protein was conducted according to the procedure described by Stellwagen et al. (22Stellwagen A.E. Haimberger Z.W. Veatch J.R. Gottschling D.E. Genes Dev. 2003; 17: 1-12Crossref PubMed Scopus (236) Google Scholar). Purification of His6-tagged Cdc13(451–693)p—The Escherichia coli expression system was used for Cdc13(451–693)p expression. Plasmids pET6H-CDC13(451–693) carrying the cdc13R635C mutation of CDC13 were constructed by ligating the 0.73-kbp BamHI-NruI cdc13R635C DNA fragment of pRS315ΔB-CDC13 with pET6H that was linearized with the same enzymes (30Lin Y.-C. Lee Y.-H.W. Lin J.-J. Biochem. J. 2007; 403: 289-295Crossref PubMed Scopus (3) Google Scholar). E. coli BL21(DE3) pLysS was used as the host for Cdc13(451–693)p expression. The purification procedure was as described previously (31Lin Y.-C. Hsu C.-L. Shih J.-W. Lin J.-J. J. Biol. Chem. 2001; 276: 24588-24593Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Purification of Recombinant Yku Proteins from Sf21 Cells—Insect cell line Sf21 was used as the host for baculovirus propagation, expression, and purification of recombinant Yku70p and Yku80p. Plasmid pBac6His-YKU70 was constructed by inserting a 1.8-kbp NcoI-SalI fragment of YKU70 to NcoI- and SalI-digested pBac6His (31Lin Y.-C. Hsu C.-L. Shih J.-W. Lin J.-J. J. Biol. Chem. 2001; 276: 24588-24593Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). Similarly, plasmid pBac6His-YKU80 was constructed by inserting a 1.9-kbp NcoI-SalI fragment of YKU80 to NcoI- and SalI-digested pBac6His. These plasmids enabled the expression of Yku70p or Yku80p with His6 tagged at the N terminus. Recombinant viruses were generated by co-transfection of plasmid pBac6His-YKU70 or pBac6His-YKU80 and Bac-N-Blue DNA to Sf21 cells (Invitrogen). To purify His6-tagged Yku70 or Yku80, ∼5 × 107 Sf21 cells were infected with 25 × 107 recombinant viruses for 4 days. Cells were washed with phosphate-buffered saline and then lysed by the addition of lysis buffer (6 m guanidine HCl, 100 mm NaH2PO4, pH 8, 10 mm Tris-HCl). The suspensions were incubated on ice for 60 min and sonicated. Total cell extracts were then collected by centrifugation. Ni-NTA-agarose (Qiagen) was utilized to purify the His6-tagged Yku70p or Yku80p. Batch purification protocol was used according to manufacturer's suggestion. The bound His6-tagged Yku70p or Yku80p protein was first washed with lysis buffer containing 20 mm imidazole and eluted by the same buffer containing 200 mm imidazole. To renature the purified Yku70p and Yku80p proteins, proteins eluted from Ni-NTA-agarose column was diluted to ∼50 μg/ml and dialyzed against renaturation buffer (100 mm Tris, pH 8.0, 2 mm EDTA, 2 mm dithiothreitol, 0.4 m l-arginine, 20% glycerol) at 4 °C for 12 h. Purified protein was aliquoted and frozen by dry ice/ethanol bath. Electrophoretic Mobility Shift Assay (EMSA)—Oligonucleotide (Table 1) was first 5′-end-labeled with [γ-32P]ATP (3000 mCi/mm; PerkinElmer Life Sciences) using T4 polynucleotide kinase (New England Biolabs) and subsequently purified from a 10% sequencing gel after electrophoresis. To prepare the duplex or tailed-duplex DNA substrates, the 5′-end-labeled DNA was mixed with an excess amount of unlabeled complementary DNA and incubated at 100 °C for 5 min. The DNA mixtures were then cooled under room temperature and subsequently separated by a native 8% polyacrylamide gel to isolate the duplex or tailed-duplex DNA substrates. To perform the assays, purified proteins were mixed with 2.0 ng of 32P-labeled DNA with a total volume of 15 μl containing 50 mm Tris-HCl, pH 7.5, 1 mm EDTA, 50 mm NaCl, and 1 mm dithiothreitol. The mixtures were allowed to incubate at room temperature for 10 min. 3 μl of 80% glycerol was then added, and the mixtures were loaded on a 6% nondenaturing polyacrylamide gel, which were pre-run at 125 V for 10 min. Electrophoresis was carried out in TBE (89 mm Tris borate, 2 mm EDTA) at 125 V for 105 min. The gels were dried and autoradiographed. Binding activity was quantified using a PhosphorImager (GE Healthcare).TABLE 1Oligonucleotides used in this study DNase I Footprint Analysis—Telomeric DNA RT1 were 5′-labeled, annealed to RT2, and purified as described above. The DNA was mixed with Yku protein in 50 μl of reaction buffer (10 mm Tris-HCl, pH 8.0, 2.5 mm MgCl2, 0.5 mm CaCl2) and incubated at room temperature for 10 min. DNase I (0.2 unit) was added and incubated at 37 °C for another 10 min. The reaction was stopped with 10 μl of 250 mm EGTA. The DNA was then precipitated at –70 °C by adding 1 μl of 10 mg/ml oyster glycogen and 150 μl of ethanol. The precipitant was collected by centrifugation, dried, and analyzed by electrophoresis using 10% polyacrylamide sequencing gel. Chromatin Immunoprecipitation Analysis on a de Novo Synthesized Telomere—Yeast strain YJL0801 was generated by introducing a CDC13-myc9::TRP1 into UCC5706 (MATa-inc ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1::GAL1-HO-LEU-2 VII-L::ADE2-TG1–3-HO site-LYS2 rad52::hisG, a gift of D. E. Gottschling (32Diede S.J. Gottschling D.E. Cell. 1999; 99: 723-733Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar)) and then transformed with plasmid pRS423TEF-KU80-TAP (22Stellwagen A.E. Haimberger Z.W. Veatch J.R. Gottschling D.E. Genes Dev. 2003; 17: 1-12Crossref PubMed Scopus (236) Google Scholar). The strain was used in chromatin immunoprecipitation experiments on de novo synthesized telomeres (32Diede S.J. Gottschling D.E. Cell. 1999; 99: 723-733Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). Briefly, yeast cells were grown in medium (2.5% raffinose) lacking histidine, tryptophan, and lysine until the cells reach the concentration of 7 × 106 cells/ml. The cells were then arrested at M phase using 10 μg/ml nocodazole at 30 °C until ∼90% of cells with the morphology of large buds. To induce HO endonuclease expression, the nocodazole-treated cells were centrifuged and resuspended in prewarmed medium containing 3% galactose and 10 μg/ml nocodazole. HO cutting efficiency was determined by Southern blotting analysis using probe near the HO site at different time points (see Fig. 7). Chromatin immunoprecipitation experiments were performed as described (33Taggart A.K.P. Teng S.-C. Zakian V.A. Science. 2002; 297: 1023-1026Crossref PubMed Scopus (302) Google Scholar), except that the TAP-tagged Ku80 was precipitated directly by IgG-Sepharose (Amersham Biosciences), and anti-Myc antibody followed by protein G-Sepharose (Sigma) was used for the Cdc13p immunoprecipitations. Quantification of the immunoprecipitated DNAs was achieved using real time PCR on a StepOne real time system (Applied Biosystems). Enrichment of DNA from the sequences inserted near the HO cut site (XIP) over an internal control DNA (XIPi) located about 47 kb from the left arm of chromosome VII was determined after normalization. Primer pairs used in amplifying XIP and XIPi were as described (34Diede S.J. Gottschling D.E. Curr. Biol. 2001; 11: 1336-1340Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). Values determined from immunoprecipitates were then normalized against values obtained from total cell extracts (input). Yku Bound to Duplex and Tailed-duplex TG1–3 DNA in Vitro— Human Ku binds to double-stranded ends in a sequence-independent manner. It was shown that the exact structure of the DNA ends does not appear to be crucial for its binding. Human Ku binds to DNA ends with 5′- or 3′-overhangs, or with hairpin loops (5Falzon M. Fewell J.W. Kuff E.L. J. Biol. Chem. 1993; 268: 10546-10552Abstract Full Text PDF PubMed Google Scholar, 35Rathmell W.K. Chu G. Mol. Cell. Biol. 1994; 14: 4741-4748Crossref PubMed Scopus (147) Google Scholar), and it was also shown to bind to tailed-duplex DNA structure formed by telomeric DNA (15Bianchi A. de Lange T. J. Biol. Chem. 1999; 274: 21223-21227Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Thus, it is interesting to test if Yku also bind to telomeric DNA in vitro. Using TAP-tagged Yku80, Yku was purified to near-homogeneity after two rounds of affinity purifications (Fig. 1A). The DNA binding activity of the purified Yku was accessed by electrophoretic mobility shift assays (EMSA). Similar to the property of mammalian Ku, the purified Yku bound to double-stranded DNA formed by two oligonucleotides with random sequences, did not bind to single-stranded DNA with the same sequences, and required a DNA end for binding (data not shown). To determine whether Yku can bind to duplex telomeric DNA, two complementary oligonucleotides with telomeric DNA sequences were synthesized and annealed (T1T2). The resulting duplex DNA has 9 bp of telomeric DNA on both ends. Yku appeared to bind efficiently to T1T2 telomeric DNA duplex (Fig. 1B). We next designed a tailed-duplex DNA substrate by annealing T3 and T4 oligonucleotides. The resulting DNA mimics the structure of telomere that has a 9-bp telomeric DNA duplex and a 15 mer-single-stranded G-tail on both ends. As shown in Fig. 1B, Yku formed complexes to the tailed-duplex DNA, and it also appeared that two complexes were formed with the DNA substrate indicating that Yku might bind to both ends of the DNA substrate. To further investigate the binding of Yku on tailed-duplex telomeric DNA, DNase I footprint experiments were conducted. The length of the duplex DNA substrate was extended to 65 bases (RT1RT2) to increase the resolution of the experiment. Results shown in Fig. 1C showed the autoradiograms of DNase I footprint analysis and the summary of the footprint results. Our results clearly indicated that Yku protein made two distinct footprints on the tailed-duplex DNA. Analysis of the locations of these two DNase I-protected regions indicated that Yku bound to both ends of the duplex DNA with protected regions of 13 and 15 bp on both ends (Fig. 1C). Because DNase I preferentially digests duplex over single-stranded DNA, the binding of Yku on single-stranded telomeric DNA cannot be determined here. Nevertheless, our results clearly indicated that Yku bound to the duplex and single-stranded junctions of the tailed duplex telomeric DNA. Using EMSA as an assay, we next determined the binding affinity of Yku to duplex DNA or tailed-duplex DNA (Table 2). For a better comparison, we have designed a set of DNA substrates with the same length (Table 1). Under our assay conditions, Yku bound to telomeric DNA duplex with an affinity similar to that of random-sequence DNA duplex (Table 2, compare T11T12 with R5R6). The addition of a 15-mer single-stranded tail with random sequence decreased slightly the binding affinity (compare R7R8 with R5R6). Interestingly, the single-stranded telomeric tail reduced the binding affinity by ∼3-fold (compare T13T14 with R5R6). Thus, our results indicated that Yku is capable of binding to tailed-duplex telomeric DNA, although with an affinity moderately lower than that of other double-stranded ends.TABLE 2Binding affinity of Yku to DNA substratesDNA substrateDescriptionLength (duplex + single strand tail)Kd,appnmR5R6Duplex18 + 053 ± 13T11T12Duplex18 + 088 ± 28R7R8Tailed-duplex18 + 1577 ± 30T13T14Tailed-duplex18 + 15186 ± 43 Open table in a new tab Sequential Formation of Ternary Complex on Telomeres by Yku and Cdc13p—Except for late S phase, a single-stranded tail with 12–14-mer TG1–3 sequence was detected in most parts of the cell cycle (25Larrivee M. LeBel C. Wellinger R.J. Genes Dev. 2004; 18: 1391-1396Crossref PubMed Scopus (189) Google Scholar). We were next interested in testing whether such a single-stranded tail is sufficient for binding by both Yku and Cdc13p. The T3T4 tailed-duplex DNA was used as the substrate in our studies. We have also expressed a His6-tagged Cdc13(451–693)p in E. coli and purified this tagged protein using Ni-NTA-agarose (Fig. 2A). The His6-tagged Cdc13(451–693)p contains the DNA binding domain and has been shown previously that it has the single-stranded telomeric DNA binding activity similar to that of full-length Cdc13p (31Lin Y.-C. Hsu C.-L. Shih J.-W. Lin J.-J. J. Biol. Chem. 2001; 276: 24588-24593Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). The result shown in Fig. 2B demonstrated that this His6-tagged Cdc13(451–693)p is capable of binding to the tailed-duplex DNA substrate (lanes 6–9). Interestingly, after initial loading of Yku, the substrate could then be bound by Cdc13(451–693)p to form several ternary complexes (Fig. 2B, lanes 11–13). Judging by the relative migrations and supershift analyses, the c1 complex may have two molecules of Yku and one molecule of Cdc13 on one DNA, whereas c2 and c3 each has one molecule of Yku and one or two molecules of Cdc13p, respectively. Thus, our DNA substrate that mimics the structure of telomere enables the formation of ternary complex by both Yku and Cdc13p. Moreover, because pre-loading of Cdc13p appeared to inhibit the binding of Yku to DNA, the result also indicated that the formation of these ternary complexes was sequential (Fig. 2B, lanes 14–17). To further demonstrate that specific binding of Cdc13p to single-stranded telomeric tail is required for the formation of ternary complex, similar experiments were conducted using DNA substrates that were either without the single-stranded tail (R1R2) or with a random-sequence single-stranded tail (R3R4). Results shown in Fig. 3 clearly indicated that a single-stranded telomeric tail is required for Cdc13p to bind and to form ternary complexes with Yku. We have previously isolated several cdc13 mutants that are defective in binding to telomeric DNA (30Lin Y.-C. Lee Y.-H.W. Lin J.-J. Biochem. J. 2007; 403: 289-295Crossref PubMed Scopus (3) Google Scholar). One of the mutants with an alternation from Arg-635 to Cys (R635C) failed to interact with single-stranded telomeric DNA. The Arg-635 was shown to be located at the DNA-binding surface of Cdc13p (36Mitton-Fry R.M. Anderson E.M. Hughes T.R. Lundblad V. Wuttke D.S. Science. 2002; 296: 145-147Crossref PubMed Scopus (158) Google Scholar). We next analyzed if the telomere binding activity of Cdc13p is required for ternary complex formation. Using the procedures similar to that in purifying wild-type protein, we have purified Cdc13(451–693)p with the R635C mutation to homogeneity (Fig. 4A). Formation of the ternary complex by Cdc13R635C mutant was then tested on T3T4 tailed-duplex DNA. The purified Cdc13R635C mutant failed to bind to the DNA substrate (Fig. 4B, lanes 5–8). It also failed to form a ternary complex with Yku and did not affect the binding of Yku to DNA. Thus, our results indicated that Cdc13p binds to the single-stranded telomeric DNA portion of the tailed-duplex DNA, and its binding activity is required for the formation of ternary complex with Yku.FIGURE 4Telomeric DNA binding activity of Cdc13(451–693)p is required for ternary complex formation. A, purification of His6-tagged Cdc13(451–693)R635Cp. Two μg of the purified protein were analyzed by 10% SDS-PAGE and stained with Coomassie Blue. B, Cdc13(451–693)R635Cp fails to form complex with Yku-DNA. Around 2 ng of 32P-labeled T3T4 DNA substrate were incubated with Yku and/or Cdc13(451–693) R635Cp and analyzed by EMSA. The amounts of Yku used in lanes 2–5 and 14–17 were 50, 150, 450, and 1350 nm, respectively. Lanes 10–13 each has 1350 nm Yku. The amounts of Cdc13(451–693)R635Cp were the same as those for Fig. 2B. An autoradiogram is shown here.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Stably Formed Yku Ring Is Required for Formation of Yku-Cdc13p-DNA Ternary Complex—Because human Ku heterodimer forms a ring structure to encircle DNA and it binds DNA through the open ends (7Walker J.R. Corpina R.A. Goldberg J. Nature. 2001; 412: 607-614Crossref PubMed Scopus (869) Google Scholar), it is reasonable to postulate that Yku interacts with telomeres in the form of a ring structure. Thus, a simple interpretation on the inhibition of Yku binding by pre-loaded Cdc13p would be Cdc13p blocking the passage of Yku ring to its target site. However, it is still possible that the pre-loaded Cdc13p occupies the DNA-binding site of Yku to prevent the binding of Yku. To rule out this possibility, we prepared recombinant Yku70 and Yku80 (rYku) separately from insect Sf21 cells using baculovirus expression systems (Fig. 5A). Although neither of these two recombinant proteins bound to blunt-end duplex DNA, the Yku activity could be readily reconstituted by mixing these two proteins (Fig. 5B). The reconstituted Yku did not bind to single-stranded DNA (Fig. 5C). The binding activity of the reconstituted Yku to the DNA ends was further confirmed by competition analysis using close circle and restriction enzyme-digested plasmid DNA. The rYku-DNA complex cannot be competed away by close circular plasmid, whereas it was readily competed away by DNAs that were digested with restriction enzymes to expose DNA ends (Fig. 5D). The degree of competition did not appear to be dependent on the type of free DNA ends generated as 3′-overhang generated by PstI competed as well as 5′-overhang generated by HindIII or blunt ends generated by PvuII. Instead, competition was increased as the number of free DNA ends increased (Fig. 5D, lane 7). Thus, the binding activity of reconstituted Yku to telomere tail-duplex DNA was specific for DNA ends, and its binding activity is similar to that of endogenous Yku protein. However, in contrast to the Yku isolated from yeast that has a half-life on DNA for >60 min, the reconstituted protein did not stably bind to DNA. It had an estimated half-life on DNA for ∼1 min (Fig. 5E). The reconstituted Yku heterodimer did not appear to form a stable ring-like structure on DNA. Although the mechanism of how reconstituted Yku is less stable on DNA is not clear to us, it is
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