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• W2076192570 abstract "Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg2+, not Ca2+, and is inhibited by Zn2+. The enzyme generates blunt ends or ends with 1-base 5′-overhangs possessing 5′-phosphate and 3′-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research. Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg2+, not Ca2+, and is inhibited by Zn2+. The enzyme generates blunt ends or ends with 1-base 5′-overhangs possessing 5′-phosphate and 3′-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research. DNA fragmentation factor caspase-activated deoxyribonuclease (also termed DFF40) caspase-activated nuclease (also termed DFF40 and CAD) 45-kDa subunit of DFF 40-kDa subunit of DFF high mobility group inhibitor of CAD (also termed DFF45) micrococcal nuclease long terminal repeat base pair(s) kilobase pair(s) Apoptosis, or programmed cell death, plays an important role in the development of an organism and in the maintenance of tissue homeostasis (reviewed in Refs. 1.Jacobson M.D. Weil M. Raff M.C. Cell. 1997; 88: 347-354Abstract Full Text Full Text PDF PubMed Scopus (2415) Google Scholar and 2.Nagata S. Cell. 1997; 88: 355-365Abstract Full Text Full Text PDF PubMed Scopus (4561) Google Scholar). Hallmarks of the terminal stages of apoptosis are nucleosomal DNA fragmentation (also termed here “DNA laddering”) and chromatin condensation (3.Wyllie A.H. Nature. 1980; 284: 555-556Crossref PubMed Scopus (4163) Google Scholar, 4.Wyllie A.H. Morris R.G. Smith A.L. Dunlop D. J. Pathol. 1984; 142: 66-77Crossref Scopus (1443) Google Scholar, 5.Compton M.M. Cancer Metast. Rev. 1992; 11: 105-119Crossref PubMed Scopus (494) Google Scholar). The endonuclease primarily responsible for mediating DNA laddering is activated by caspase-3 treatment of DFF.1 In its inactive form, DFF is a heterodimer composed of a 45-kDa chaperone and inhibitor subunit (DFF45/ICAD) and a 40-kDa latent endonuclease subunit (DFF40/CAD/CPAN) (6.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 7.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 8.Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar, 9.Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar, 10.Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar). This protein complex resides in the cell nucleus (7.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 11.Samejima K. Earnshaw W.C. Exp. Cell Res. 1998; 243: 453-459Crossref PubMed Scopus (45) Google Scholar). Caspase-3 cleavage of DFF specifically cuts only DFF45, which results in the dissociation of cleaved DFF45 from DFF40 (6.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 7.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 8.Enari M. Sakahira H. Yokoyama H. Okawa K. Iwamatsu A. Nagata S. Nature. 1998; 391: 43-50Crossref PubMed Scopus (2812) Google Scholar, 9.Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar, 10.Halenbeck R. MacDonald H. Roulston A. Chen T.T. Conroy L. Williams L.T. Curr. Biol. 1998; 8: 537-540Abstract Full Text Full Text PDF PubMed Google Scholar). Interestingly, the released endonuclease forms homo-oligomers that are the enzymatically active form of DFF40 (12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). In addition, its activity on naked DNA substrates can be further activated by specific chromosomal proteins, such as histone H1 or HMG-1/2 (7.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar, 12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar, 13.Toh S.Y. Wang X. Li P. Biochem. Biophys. Res. Commun. 1998; 250: 598-601Crossref PubMed Scopus (40) Google Scholar), and topoisomerase II (this report). Furthermore, chromatin condensation can be initiated in isolated nuclei by the addition of recombinant activated DFF40, purified free of caspase-3 and DFF45 breakdown products (7.Liu X. Li P. Widlak P. Zou H. Luo X. Garrard W.T. Wang X. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8461-8466Crossref PubMed Scopus (502) Google Scholar). It should be noted that the physiological significance of DFF in triggering DNA laddering and chromatin condensation during apoptosis has been unequivocally proven. A homozygous deletion of the single copy gene encoding DFF45 has been created in the mouse germ line (14.Zhang J. Liu X. Scherer D.C. Kaer L.V. Wang X. Xu M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12,480-12,485Crossref Scopus (159) Google Scholar). Significantly, thymocytes and splenocytes from these knockout mice exhibit greatly reduced DNA laddering or chromatin condensation when exposed to apoptotic stimuli, both in vivo and in vitro, proving that DFF is a principal player for the induction of these events (14.Zhang J. Liu X. Scherer D.C. Kaer L.V. Wang X. Xu M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12,480-12,485Crossref Scopus (159) Google Scholar, 15.Zhang, J., Wang, X., Bove, K. E., and Xu, M. (1999) J. Biol. Chem.(In Press)Google Scholar). Furthermore, such mice still express DFF40, indicating that the chaperone function of DFF45 is required in vivo for creating a potentially functional DFF40 endonuclease. 2M. Xu, personal communication. Other gene products also appear to participate in the DFF pathway. Two proteins encoded by genes called cell death-inducing DFF45-like effectors, or CIDEs, which exhibit homology to the N-terminal domain of DFF45, can activate apoptosis in a DFF45-inhibitable fashion, but their precise mechanism of action remains to be elucidated (16.Inohara N. Koseki T. Chen S. Wu X. Nunez G. EMBO J. 1998; 17: 2526-2533Crossref PubMed Scopus (283) Google Scholar). In addition, an isoform of DFF45, termed DFF35 (also termed ICAD-S (9.Sakahira H. Enari M. Nagata S. Nature. 1998; 391: 96-99Crossref PubMed Scopus (1427) Google Scholar)), is incapable of acting as a chaperone and acts as an inhibitor of the latent endonuclease (17.Sakahira H. Enari M. Nagata S. J. Biol. Chem. 1999; 274: 15,740-15,744Abstract Full Text Full Text PDF Scopus (88) Google Scholar, 18.Gu J. Dong R.-P. Zhang C. McLaughlin D.F. Wu M.X. Schlossman S.F. J. Biol. Chem. 1999; 274: 20,759-20,762Abstract Full Text Full Text PDF Scopus (75) Google Scholar). In conclusion, it is well established experimentally that DFF plays a major and regulated role in apoptotic DNA fragmentation and chromatin condensation, although other pathways have also been identified (19.Susin S.A. Lorenzo H.K. Zamzami N. Marzo I. Snow B.E. Brothers G.M. Mangion J. Jacotot E. Costantini P. Loeffler M. Larochette N. Goodlett D.R. Aebersold R. Siderovski D.P. Penninger J.M. Kroemer G. Nature. 1999; 397: 441-446Crossref PubMed Scopus (3464) Google Scholar, 20.Sakahira H. Enari M. Ossawa Y. Uchiyama Y. Nagata S. Curr. Biol. 1999; 9: 543-546Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Nucleases have proven to be valuable reagents for the analysis of chromatin structure (21.van Holde K.E. Chromatin. Springer, Berlin1988Google Scholar). Because apoptotic nucleases are known to efficiently create DNA ladders composed of total genomic sequences, if DFF40 is the major player in such laddering, then its sequence specificity for DNA cleavage would appear to be broad enough to be a useful additional tool for chromatin research. Here we report the cleavage site preferences for DFF40 on naked DNA and chromatin substrates and demonstrate that DFF40 is indeed a useful reagent for generating sharp internucleosomal DNA cleavage. HeLa cell nuclei were purified as described elsewhere (6.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar). Nuclei were successively washed at 4 °C in 3 mm MgCl2, 10 mm KCl, 1 mm dithiothreitol, 20 mm Tris, pH 7.6, 0.2m sucrose (buffer A) plus 1% Triton X-100 and then in buffer A alone, suspended at 1 μg/μl as DNA in buffer A plus 20%, glycerol and aliquots were stored at −80 °C. Chromatin was reconstituted on a tandem 18-mer array of the Lytechinus variegatus 5 S rRNA gene (22.Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Abstract Full Text PDF PubMed Scopus (374) Google Scholar), recloned into the vector pGEM3A to yield the recombinant plasmid pGH 207-18 (gift of Dr. Joe Gatewood, Los Alamos National Laboratory). The 18-mer was excised from pGH 207-18 by digestion with HhaI and purified from agarose gels after electrophoresis. Chromatin was reconstituted using a salt step dialysis procedure (23.Rhodes D. Laskey R.A. Methods Enzymol. 1989; 170: 575-585Crossref PubMed Scopus (63) Google Scholar, 24.Stein A. Methods Enzymol. 1989; 170: 585-603Crossref PubMed Scopus (64) Google Scholar). Core histones were purified from HeLa nuclei. Briefly, nuclei were incubated with MNase (Worthington) and washed with buffer containing 1 mm EGTA, and chromatin was eluted with 2 mm EDTA. Chromatin was equilibrated with 0.4m NaCl and loaded onto a hydroxyapatite column. After washing the column extensively with 0.4 m NaCl, histone H1 was eluted with 0.6 m NaCl, and core histones were subsequently eluted with 2 m NaCl (25.Simon R. Felsenfeld G. Nucleic Acids Res. 1979; 6: 689-696Crossref PubMed Scopus (293) Google Scholar); no other protein bands were visible on overloaded SDS-polyacrylamide gels. Core histones were mixed with DNA at a histone:DNA weight ratio of 1.4 in 2.5m NaCl, 20 mm Tris, 1 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, pH 7.6. The mixture was incubated for 15 min at 37 °C, added to a dialysis tube (SpectraPor;M r cut-off of 6000–8000) and dialyzed at 4 °C as follows: 2 h with 1 m NaCl, 3 h with 0.55 m NaCl, 4 h with 0.25 m NaCl, and finally 12 h with buffer without NaCl. If histone H1 was also to be assembled into chromatin, histone H1 purified from HeLa cell nuclei as described above was added at the 0.55 m NaCl step at a concentration of 0.28 μg/μg of DNA. Chromatin was concentrated using Millipore Ultrafree-MC spin filters to about 0.25 μg of DNA/μl and stored at −80 °C after glycerol was added to a final concentration of 10%. Recombinant caspase-3 was prepared as described (12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). DFF was either purified from HeLa cells or from an Escherichia coli expression system as reported previously (6.Liu X. Zou H. Slaughter C. Wang X. Cell. 1997; 89: 175-184Abstract Full Text Full Text PDF PubMed Scopus (1650) Google Scholar, 12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Similar results were obtained with either source of the protein. However, the recombinant protein had a significantly lower specific activity, possibly due to a lack of the appropriate post-translational modifications. One μg of naked DNA, nuclei, or chromatin (as DNA) was incubated at 37 °C with 1 μl of caspase-3 (0.2 μg) and 1 μl of DFF (1 unit) in buffer consisting of 10 mm KCl, 3 mm MgCl2, 0.5 mm dithiothreitol, 10 mm Hepes, pH 7.5 (final volume 15 μl) for varying times, in the absence or presence of supplemented proteins as indicated in the legends to Figs. 3, 4, and 6. When comparisons were made between DFF and MNase, unless otherwise indicated, 3 mm CaCl2 was added to the above buffer for the MNase reactions. As controls, we routinely preincubated caspase-3 with 10 μm Ac-DEAD-CHO (a tetrapeptide aldehyde inhibitor of caspase-3) to demonstrate caspase-3 dependence on DFF activation or similarly postincubated with the inhibitor to block any further action of the protease before the addition of activated DFF to any of the above substrates. Although we have found that digestion of naked DNA by DFF was markedly more active under conditions of higher monovalent cations (e.g. 50–100 mm), this is not true for digestion of chromatin substrates; thus, to reduce potential protein exchange and redistribution during digestion of chromatin, we chose to employ buffers containing 10–25 mmmonovalent cations. Aliquots of the endonuclease reaction were mixed with ½ volume of stop solution (0.6% SDS, 50 mmEDTA, and 6 mg/ml proteinase K) and incubated for 1 h at 50 °C. Gel loading dye buffer was added, and samples were then run on 1.5% SeaKem agarose gels using 1× TAE as the running buffer. After electrophoresis, DNA was stained with ethidium bromide, and gels were scanned with a FluorImager (Molecular Dynamics Inc., Sunnyvale, CA). Images were analyzed using ImageQuant software (Molecular Dynamics) and have been represented as negatives. When radioactive DNA was used as the substrate, gels were dried and imaged with a PhosphorImager (Molecular Dynamics), and images were analyzed as above.Figure 4Cleavage of reconstituted chromatin by caspase-3-activated DFF. An 18-mer of the 5 S rRNA gene, as naked DNA or in vitro reconstituted chromatin, was incubated in the presence of caspase-3 and DFF for 5, 10, 20, and 40 min at 37 °C. As a control, the reconstituted chromatin was also separately incubated with MNase (0.1 units/μl, with 3 mmCaCl2) for 1, 3, 10, and 30 min at 25 °C. DNA digestion products were purified and resolved on an agarose gel, and DNA was visualized after staining with ethidium bromide. M, molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 6Activation of DFF cleavage by chromosomal proteins on naked DNA and chromatin substrates. A, 1 μg of 5 S rDNA 18-mer naked DNA or in vitro reconstituted chromatin were incubated at 37 °C for 15 min with caspase-3-activated DFF and bovine serum albumin (200 ng), HeLa cell histone H1 (100 ng), human HMG-1 (200 ng) (gift of Michael Bustin), or human topoisomerase II (Topo II; 1 unit; Topogen) as indicated. DNA digestion products were purified and resolved on an agarose gel, and DNA was visualized after staining with ethidium bromide. B, 1 μg of 5 S rDNA 18-mer in vitroreconstituted core histone-containing or core histone plus histone H1-containing chromatin were incubated at 25 °C with 1 unit of MNase for 1, 3, 10, and 30 min or at 37 °C with caspase-3 activated DFF for 5, 10, 20, and 40 min, as indicated. DNA digestion products were purified and resolved on an agarose gel, and DNA was visualized after staining with ethidium bromide.View Large Image Figure ViewerDownload Hi-res image Download (PPT) For a relative comparison of DNA cleavage site sequence preferences between different endonucleases, pUC19 DNA was digested with EcoRI,32P-5′-end-labeled with T4 polynucleotide kinase after dephosphorylation, and then DNA circles were created in a T4 DNA ligase-catalyzed reaction as described previously (26.Widlak P. Gaynor R.B. Garrard W.T. J. Biol. Chem. 1997; 272: 17654-17661Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Resulting DNA circles (500 ng) were incubated with 0.25 units of MNase (Worthington) or with 0.0025 units of DNase I (Worthington) for 1, 2, 4, and 6 min, or with 0.5 units of DFF with 0.2 μg of caspase-3 for 3, 9, 30, and 60 min at 37 °C. Reaction mixtures were 10 μl each and contained MgCl2 and CaCl2 at 1.5 mm each. DNA was purified, digested with AvaI, and separated on 5% polyacrylamide sequencing gels. For detailed analyses of sequences at cleavage sites, a 177-bp fragment of the HIV-1 5′-LTR DNA was excised from plasmid pWLTR11 (26.Widlak P. Gaynor R.B. Garrard W.T. J. Biol. Chem. 1997; 272: 17654-17661Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar) with BglII and AvaI, purified from an agarose gel after electrophoresis, and 5′-end-labeled with T4 polynucleotide kinase. Labeled DNA was incubated with caspase-3 and DFF for 10 min at 37 °C, and then purified by phenol/chloroform extraction and ethanol precipitation. To analyze cleavage sites on the coding strand, DNA was digested with BsaI (releasing a 12-bp fragment labeled at the BglII site and allowing direct analysis from the AvaI site). To analyze cleavage sites on the noncoding strand, DNA was digested with ScaI (releasing a 20-bp fragment labeled at the AvaI site and allowing direct analysis from the BglII site). Digestion products were resolved on 6% polyacrylamide sequencing gels together with the appropriate Sanger sequencing reactions (DNA was sequenced with the Amersham T7 Sequenase version 2.0 sequencing kit according to the vendor's protocol). Reconstituted chromatin was incubated with caspase-3 and DFF, and then DNA was purified. Mononucleosomal DNA was isolated after electrophoresis on a low melting agarose gel, 5′-end-dephosphorylated with shrimp alkaline phosphatase (Roche Molecular Biochemicals), and 32P-5′-end labeled with T4 polynucleotide kinase. Aliquots of labeled DNA were digested withEcoRI or 3′-end modified with either T4 DNA polymerase (U. S. Biochemical Corp.) or terminal deoxynucleotidyl transferase (U. S. Biochemical Corp.) according to the vendor's protocols. DNA samples were then resolved on 6% polyacrylamide sequencing gels. A variety of endonucleases have been implicated in apoptotic DNA laddering, including non-metal ion-dependent (27.Fernandes R.S. Cotter T.G. 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Cidlowski J.A. J. Biol. Chem. 1997; 272: 6677-6884Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). To rigorously establish the divalent ion requirements for DFF, we first dialyzed the protein against a buffer containing EDTA. We found that caspase-3-activated DFF endonuclease had a pH optimum of 7.5 and only required Mg2+ and not Ca2+ as its divalent cation, even in the presence of EGTA, thereby eliminating the possibility that traces of contaminating Ca2+ in Mg2+-containing solutions may be required for its activity (data not shown) (see Ref. 42.Price P.A. J. Biol. Chem. 1975; 250: 1981-1986Abstract Full Text PDF PubMed Google Scholar). Under the optimal reaction conditions, the enzyme does not digest either single-stranded DNA or RNA (data not shown). Because Zn2+has been reported to block apoptotic DNA laddering in certain systems (43.Ojcius D.M. Zychlinsky A. Zheng L.M. Young J.D.-E. Exp. Cell. Res. 1991; 197: 43-49Crossref PubMed Scopus (226) Google Scholar, 44.Sun D.Y. Jiang S. Zheng L.-M. Ojcius D.M. Young J.D.-E. J. Exp. Med. 1994; 179: 559-568Crossref PubMed Scopus (89) Google Scholar), we also tested this ion and found it to be a strong inhibitor of DFF endonuclease activity (data not shown). DFF potentially may be a useful reagent for chromatin structural analyses. We therefore evaluated DFF's sequence preferences for naked DNA cleavage in comparison with those of MNase and DNase I, which have both been well characterized previously (45.Hörz W. Altenburger W. Nucleic Acids Res. 1981; 9: 2643-2658Crossref PubMed Scopus (238) Google Scholar, 46.Dingwall C. Lomonossoff G.P. Laskey R.A. Nucleic Acids Res. 1981; 9: 2659-2673Crossref PubMed Scopus (253) Google Scholar, 47.Drew H.R. Travers A.A. Cell. 1984; 37: 491-502Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 48.Drew H.R. J. Mol. Biol. 1984; 176: 535-557Crossref PubMed Scopus (226) Google Scholar). Separation of the corresponding cleavage products of 32P-5′-labeled pUC19 DNA on a sequencing gel reveals that caspase-3-activated DFF endonuclease possesses moderately more sequence selectivity than either MNase or DNase I (Fig. 1). Nevertheless, DNA products are eventually processed to fragments ≤20 bp after more extensive digestion (data not shown), indicating a quite broad sequence specificity, consistent with the fact that DFF endonuclease is capable of converting purified HeLa cell DNA quantitatively into very small DNA fragments (see below). We determined the nucleotide sequences at various cleavage sites by using 32P-end-labeled DNA restriction fragments as substrates and resolution of the Watson or Crick strands on sequencing gels. As shown in Fig. 2, the endonuclease generates blunt ends or ends with 1-base 5′-overhangs. We analyzed 57 cleavage sites at the nucleotide level within the HIV-1 LTR, the mouse immunoglobulin κ gene, and pUC19 DNA. While these analyses revealed no simple consensus cleavage sequence, there clearly was a preference with respect to purines and pyrimidines. The frequencies of the 4 bases found on each side of these cleavage sites were as follows: 5′-R (72%), R (74%), R (66%), Y (61%) ↓ R (65%), Y (67%), Y (75%), Y (75%)-3′. It is significant that this purine/pyrimidine preference exhibits a rotational (dyad) symmetry. This observation, together with the double-stranded cleavage activity of this enzyme, is consistent with our observation that only homo-oligomeric forms of DFF40 are enzymatically active (12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). To determine the utility of DFF as a reagent for chromatin research, we compared its action with that of MNase for generating oligonucleosomal DNA ladders. As shown in Fig. 3, digestion of chromatin in isolated HeLa cell nuclei was dependent on the presence of either enzyme (compare lanes 1 and 2 withlanes 4–7 and 9–12) and for DFF required caspase-3 cleavage (compare lanes 14–16with lanes 9–12). The average nucleosomal DNA repeat length estimated after digestion by either enzyme was approximately 180 bp, in reasonable agreement with previous reports (21.van Holde K.E. Chromatin. Springer, Berlin1988Google Scholar). Quantitation by PhosphorImager analysis revealed that 70% of the total genomic DNA could be processed to mononucleosomes by DFF after extensive digestion (Fig. 3 A, lane 9). This incompleteness was not due to DFF-resistant sequences in the human genome, because high molecular weight naked HeLa cell DNA could be quantitatively converted to very small DNA fragments upon digestion by the enzyme (Fig. 3 B, lanes 2–6). The incomplete conversion to mononucleosomes may be due to a subfraction of the genome being organized into DFF-resistant chromatin structures, and/or to nuclear heterogeneity in DFF permeability; DFF forms very large oligomeric complexes upon activation (12.Liu X. Zou H. Widlak P. Garrard W. Wang X. J. Biol. Chem. 1999; 274: 13836-13840Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), and its nuclear access may be partially limited. Consistent with this view is that the addition of 1 mm GTP to reaction mixtures stimulated the digestion kinetics about 2-fold on the nuclear substrate but had no affect on naked DNA digestion kinetics (data not shown), presumably by enhancing the nuclear import and/or retention of DFF by phosphorylation. Digestion of nuclei with caspase-3-activated DFF resulted in oligonucleosomal DNA ladders somewhat sharper than those generated by MNase, as judged by oligonucleosomal multimer band sharpness and the ability to visualize bands of longer oligomers. This is because the interband background between successive oligonucleosomal multimers was higher for MNase digestion products; unlike MNase, DDF lacks exonuclease activity. Furthermore, cleavage within nucleosome core particles was undetectable for DFF digestion products, in contrast to MNase digestion products, which exhibited subnucleosomal DNA fragments (Fig. 3 A, compare lanes 6and 7 with lanes 9 and 10; Fig. 3 C, compare lanes 1 and3). We conclude that DFF is another reagent useful for chromatin research. In order to establish an in vitro system to study further the action of DFF endonuclease on chromatin, we chose as a model substrate for in vitro chromatin assembly an 18-mer of a 207-bp 5 S rRNA gene sequence, which has been previously shown by Simpson and co-workers (22.Simpson R.T. Thoma F. Brubaker J.M. Cell. 1985; 42: 799-808Abstract Full Text PDF PubMed Scopus (374) Google Scholar) to position nucleosomes on each tandem repeat. We then used a salt step gradient procedure with purified HeLa cell core histones to reconstitute nucleosomes onto this 5 S gene tandem array (see “Experimental Procedures”). As shown in Fig.4, both DFF endonuclease and MNase generated nucleosomal DNA ladders upon digestion of the reconstituted chromatin. However, just as in the nuclear chromatin digests (Fig. 3), it is significant that the nucleosomal DNA ladders generated by DFF endonuclease are sharper than those generated by MNase. Therefore, DFF may be better than MNase as a reagent for some types of chromatin structural analyses. As expected, digestion of the naked DNA control only generated a nondiscrete smear of DNA fragmentation products (Fig.4). Interestingly, the kinetics of DNA fragmentation by DFF endonuclease on naked DNA are only severalfold faster than on the chromatin substrate (as judged by the intensities of the high molecular weight doublet bands) (see also Fig. 6), whereas we note that MNase or DNase I attack naked DNA much more rapidly than chromatin (data not shown). We interpret this result as follows. The assembly of DNA into nucleosome core particles, which would be expected to reduce DNA cleavage accessibility, is compensated by an activation of DFF endonuclease by nucleosomal DNA wrapping" @default.
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• W2076192570 date "2000-03-01" @default.
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