The dynamic structure of individual nucleosomes was examined by stretching nucleosomal arrays with a feedback-enhanced optical trap. Forced disassembly of each nucleosome occurred in three stages. Analysis of the data using a simple worm-like chain model yields 76 bp of DNA released from the histone core at low stretching force. Subsequently, 80 bp are released at higher forces in two stages: full extension of DNA with histones bound, followed by detachment of histones. When arrays were relaxed before the dissociated state was reached, nucleosomes were able to reassemble and to repeat the disassembly process. The kinetic parameters for nucleosome disassembly also have been determined.
Adrenal medullary chromaffin cells release catecholamines and neuropeptides in an activity-dependent manner controlled by the sympathetic nervous system. Under basal sympathetic tone, catecholamines are preferentially secreted. During acute stress, increased sympathetic firing evokes release of both catecholamines as well as neuropeptides. Both signalling molecules are co-packaged in the same large dense core granules, thus release of neuropeptide transmitters must be regulated after granule fusion with the cell surface. Previous work has indicated this may be achieved through a size-exclusion mechanism whereby, under basal sympathetic firing, the catecholamines are selectively released through a restricted fusion pore, while less-soluble neuropeptides are left behind in the dense core. Only under the elevated firing experienced during the sympathetic stress response do the granules fully collapse to expel catecholamines and neuropeptides. However, mechanistic description and physiological regulation of this process remain to be determined. We employ electrochemical amperometry, fluid-phase dye uptake and electrophysiological capacitance noise analysis to probe the fusion intermediate in mouse chromaffin cells under physiological electrical stimulation. We show that basal firing rates result in the selective release of catecholamines through an Omega-form 'kiss and run' fusion event characterized by a narrow fusion pore. Increased firing raises calcium levels and activates protein kinase C, which then promotes fusion pore dilation until full granule collapse occurs. Our results demonstrate that the transition between 'kiss and run' and 'full collapse' exocytosis serves a vital physiological regulation in neuroendocrine chromaffin cells and help effect a proper acute stress response.
Protein–protein interactions (PPIs) are intriguing targets in drug discovery and development. Peptides are well suited to target PPIs, which typically present with large surface areas lacking distinct features and deep binding pockets. To improve binding interactions to these topologies by PPI-focused therapeutics and advance their development, potential ligands can be equipped with electrophilic groups to enable binding through covalent mechanisms of action. We report a strategy termed electrophile scanning to identify reactivity hotspots in a known peptide ligand. Cysteine mutants of the ligand are used to install protein-reactive modifiers via a palladium oxidative addition complex (Pd-OAC). Reactivity hotspots are revealed by cross-linking reactions with the target protein under physiological conditions. In a system with the 9-mer peptide antigen VL9 and MHC class I receptor HLA-E, we identify two reactivity hotspots that afford up to 87% conversion to the protein–peptide conjugate within 4 hours. The reactions are specific to the target protein in vitro and dependent on the peptide sequence. Moreover, the cross-linked peptide successfully inhibits molecular recognition of HLA-E by CD94─NKG2A possibly due to structural changes enacted at the PPI interface. The results illustrate the potential of electrophile scanning as a tool for rapid discovery and development of covalent peptide binders.
Nucleosomes inhibit DNA repair in vitro, suggesting that chromatin remodeling activities might be required for efficient repair in vivo. To investigate how structural and dynamic properties of nucleosomes affect damage recognition and processing, we investigated repair of UV lesions by photolyase on a nucleosome positioned at one end of a 226-bp-long DNA fragment. Repair was slow in the nucleosome but efficient outside. No disruption or movement of the nucleosome was observed after UV irradiation and during repair. However, incubation with the nucleosome remodeling complex SWI/SNF and ATP altered the conformation of nucleosomal DNA as judged by UV photo-footprinting and promoted more homogeneous repair. Incubation with yISW2 and ATP moved the nucleosome to a more central position, thereby altering the repair pattern. This is the first demonstration that two different chromatin remodeling complexes can act on UV-damaged nucleosomes and modulate repair. Similar activities might relieve the inhibitory effect of nucleosomes on DNA repair processes in living cells. Nucleosomes inhibit DNA repair in vitro, suggesting that chromatin remodeling activities might be required for efficient repair in vivo. To investigate how structural and dynamic properties of nucleosomes affect damage recognition and processing, we investigated repair of UV lesions by photolyase on a nucleosome positioned at one end of a 226-bp-long DNA fragment. Repair was slow in the nucleosome but efficient outside. No disruption or movement of the nucleosome was observed after UV irradiation and during repair. However, incubation with the nucleosome remodeling complex SWI/SNF and ATP altered the conformation of nucleosomal DNA as judged by UV photo-footprinting and promoted more homogeneous repair. Incubation with yISW2 and ATP moved the nucleosome to a more central position, thereby altering the repair pattern. This is the first demonstration that two different chromatin remodeling complexes can act on UV-damaged nucleosomes and modulate repair. Similar activities might relieve the inhibitory effect of nucleosomes on DNA repair processes in living cells. cyclobutane pyrimidine dimers nucleotide excision repair T4-endonuclease V Cockayne's syndrome B multiplicity of infection ATP-using chromatin assembly factor yeast Folding of eukaryotic DNA into nucleosomes and higher order structures restricts its accessibility to proteins, thereby repressing DNA-dependent processes like transcription and DNA repair. Dynamic properties of nucleosomes and chromatin remodeling activities contribute to relieve the repressive role of chromatin. Although remodeling has been extensively studied in the context of transcriptional regulation, its contribution to DNA repair remains unclear (1Green C.M. Almouzni G. EMBO Rep. 2002; 3: 28-33Crossref PubMed Scopus (179) Google Scholar, 2Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar, 3Fyodorov D.V. Kadonaga J.T. Cell. 2001; 106: 523-525Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 4Thoma F. EMBO J. 1999; 18: 6585-6598Crossref PubMed Google Scholar, 5Smerdon M.J. Conconi A. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 227-255Crossref PubMed Scopus (117) Google Scholar). Here we show that two different nucleosome remodeling activities can act on UV-damaged nucleosomes and modulate repair by photolyase. The basic unit of chromatin is the nucleosome core particle. It consists of 147 bp of DNA wrapped in 1.65 left-handed superhelical turns around an octamer of core histones. The octamer itself consists of a histone (H3–H4)2 tetramer, which binds the central six turns of DNA, and two H2A–H2B dimers, which primarily bind distal regions of the core DNA. The structure of DNA changes upon folding into nucleosomes (6Luger K. Mader A.W. Richmond R.K. Sargent D.F. Richmond T.J. Nature. 1997; 389: 251-260Crossref PubMed Scopus (6991) Google Scholar). In principle, all reactions that involve DNA can be regulated by changing DNA packaging. A contribution to the regulation of DNA accessibility comes from intrinsic properties of nucleosomes, such as nucleosome mobility, unfolding, or partial disruption (7Widom J. Annu. Rev. Biophys. Biomol. Struct. 1998; 27: 285-327Crossref PubMed Scopus (250) Google Scholar). Another contribution is made by protein complexes that remodel chromatin structures. One class consists of histone-modifying complexes that add or remove covalent modifications from histone tails. Another class utilizes the energy of ATP hydrolysis to modify chromatin structure in a non-covalent manner (2Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar, 3Fyodorov D.V. Kadonaga J.T. Cell. 2001; 106: 523-525Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 8Peterson C.L. FEBS Lett. 2000; 476: 68-72Crossref PubMed Scopus (50) Google Scholar, 9Vignali M. Hassan A.H. Neely K.E. Workman J.L. Mol. Cell. Biol. 2000; 20: 1899-1910Crossref PubMed Scopus (595) Google Scholar). The ATP-dependent chromatin remodeling complexes contain an ATPase subunit that belongs to the SNF2 superfamily of proteins. Based on the identity of this subunit, they have been divided into the SWI2/SNF2 family, the ISWI family, and the Mi-2 family (10Boyer L.A. Logie C. Bonte E. Becker P.B. Wade P.A. Wolffe A.P. Wu C. Imbalzano A.N. Peterson C.L. J. Biol. Chem. 2000; 275: 18864-18870Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Although functional analysis of these complexes has been focused mainly on transcription, there is increasing evidence that similar activities may assist recombination and repair (1Green C.M. Almouzni G. EMBO Rep. 2002; 3: 28-33Crossref PubMed Scopus (179) Google Scholar, 2Narlikar G.J. Fan H.Y. Kingston R.E. Cell. 2002; 108: 475-487Abstract Full Text Full Text PDF PubMed Scopus (1256) Google Scholar, 3Fyodorov D.V. Kadonaga J.T. Cell. 2001; 106: 523-525Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 11Frit P. Kwon K. Coin F. Auriol J. Dubaele S. Salles B. Egly J.M. Mol. Cell. 2002; 10: 1391-1401Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 12Alexiadis V. Kadonaga J.T. Genes Dev. 2002; 16: 2767-2771Crossref PubMed Scopus (133) Google Scholar, 13Hara R. Sancar A. Mol. Cell. Biol. 2002; 22: 6779-6787Crossref PubMed Scopus (129) Google Scholar). Cyclobutane pyrimidine dimers (CPDs)1 are the major class of DNA lesions produced by UV light. The recent crystal structure of a CPD in DNA showed that the overall helical axis bends ∼30° toward the major groove and unwinds ∼9° (14Park H. Zhang K. Ren Y. Nadji S. Sinha N. Taylor J.S. Kang C. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 15965-15970Crossref PubMed Scopus (199) Google Scholar). CPDs appear to have only minimal effect on nucleosome stability but may affect nucleosome positioning during chromatin assembly. CPD formation is modulated by the structure of nucleosomes and by other protein-DNA interactions (4Thoma F. EMBO J. 1999; 18: 6585-6598Crossref PubMed Google Scholar,5Smerdon M.J. Conconi A. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 227-255Crossref PubMed Scopus (117) Google Scholar). In most organisms, pyrimidine dimers are removed by the multistep nucleotide excision repair (NER) pathway (15Hoeijmakers J.H. Nature. 2001; 411: 366-374Crossref PubMed Scopus (3164) Google Scholar). As an alternative or additional pathway, a wide variety of organisms can specifically revert photoproducts to their native bases by DNA photolyase in the presence of light (photoreactivation) (16Sancar G.B. Mutat. Res. 2000; 451: 25-37Crossref PubMed Scopus (100) Google Scholar). Both DNA repair mechanisms are modulated by chromatin structure (4Thoma F. EMBO J. 1999; 18: 6585-6598Crossref PubMed Google Scholar, 5Smerdon M.J. Conconi A. Prog. Nucleic Acids Res. Mol. Biol. 1999; 62: 227-255Crossref PubMed Scopus (117) Google Scholar). Nucleosomes exert a repressive role on NER and photoreactivation because repair is slow in regions of positioned nucleosomes and fast in linker DNA of yeast (17Wellinger R.E. Thoma F. EMBO J. 1997; 16: 5046-5056Crossref PubMed Scopus (127) Google Scholar, 18Suter B. Livingstone-Zatchej M. Thoma F. EMBO J. 1997; 16: 2150-2160Crossref PubMed Scopus (69) Google Scholar, 19Tijsterman M. de Pril R. Tasseron-de Jong J.G. Brouwer J. Mol. Cell. Biol. 1999; 19: 934-940Crossref PubMed Scopus (58) Google Scholar). However, the repression is not tight, since all lesions need to be repaired to prevent mutagenesis. In vitro, reconstituted nucleosomes also exert an inhibitory effect on repair by photolyase or T4-endonuclease V (20Schieferstein U. Thoma F. EMBO J. 1998; 17: 306-316Crossref PubMed Scopus (45) Google Scholar, 21Kosmoski J.V. Smerdon M.J. Biochemistry. 1999; 38: 9485-9494Crossref PubMed Scopus (47) Google Scholar) and by NER (13Hara R. Sancar A. Mol. Cell. Biol. 2002; 22: 6779-6787Crossref PubMed Scopus (129) Google Scholar,22Wang Z.G. Wu X.H. Friedberg E.C. J. Biol. Chem. 1991; 266: 22472-22478Abstract Full Text PDF PubMed Google Scholar, 23Liu X. Smerdon M.J. J. Biol. Chem. 2000; 275: 23729-23735Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 24Hara R. Mo J. Sancar A. Mol. Cell. Biol. 2000; 20: 9173-9181Crossref PubMed Scopus (151) Google Scholar, 25Ura K. Araki M. Saeki H. Masutani C. Ito T. Iwai S. Mizukoshi T. Kaneda Y. Hanaoka F. EMBO J. 2001; 20: 2004-2014Crossref PubMed Scopus (155) Google Scholar). Repair of SV40 minichromosomes by NER was also reduced compared with naked DNA (26Araki M. Masutani C. Maekawa T. Watanabe Y. Yamada A. Kusumoto R. Sakai D. Sugasawa K. Ohkuma Y. Hanaoka F. Mutat. Res. 2000; 459: 147-160Crossref PubMed Scopus (31) Google Scholar). However, Xenopus extracts proficient for NER can repair a lesion placed in a reconstituted nucleosome (27Kosmoski J.V. Ackerman E.J. Smerdon M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10113-10118Crossref PubMed Scopus (44) Google Scholar), suggesting that factors responsible for modulating nucleosome structure may be present in the extract. Several proteins that are involved in DNA repair pathways belong to the SNF2 superfamily of ATPases and thereby might provide chromatin remodeling activities to facilitate DNA repair (1Green C.M. Almouzni G. EMBO Rep. 2002; 3: 28-33Crossref PubMed Scopus (179) Google Scholar, 28Eisen J.A. Sweder K.S. Hanawalt P.C. Nucleic Acids Res. 1995; 23: 2715-2723Crossref PubMed Scopus (619) Google Scholar). Nucleosome remodeling activity was shown on undamaged nucleosomes for the Cockayne's syndrome B (CSB) protein, which is involved in transcription coupled repair (29Citterio E. Van Den Boom V. Schnitzler G. Kanaar R. Bonte E. Kingston R.E. Hoeijmakers J.H. Vermeulen W. Mol. Cell. Biol. 2000; 20: 7643-7653Crossref PubMed Scopus (305) Google Scholar), and for the INO80 chromatin remodeling complex of yeast. Mutations in INO80 cause sensitivity to hydroxyurea, methyl methanesulfonate, and ultraviolet and ionizing radiation (30Shen X. Mizuguchi G. Hamiche A. Wu C. Nature. 2000; 406: 541-544Crossref PubMed Scopus (667) Google Scholar). On the other hand, the ACF remodeling complex was reported to facilitate NER of a lesion placed in linker DNA of a dinucleosome but not of a lesion in the nucleosomes (25Ura K. Araki M. Saeki H. Masutani C. Ito T. Iwai S. Mizukoshi T. Kaneda Y. Hanaoka F. EMBO J. 2001; 20: 2004-2014Crossref PubMed Scopus (155) Google Scholar). In contrast to ACF, the SWI/SNF remodeling complex was most recently reported to stimulate excision nuclease activity on a nucleosome containing a bulky acetylaminofluorene-guanine adduct (13Hara R. Sancar A. Mol. Cell. Biol. 2002; 22: 6779-6787Crossref PubMed Scopus (129) Google Scholar). Thus, SWI/SNF and ACF might act differently on damaged nucleosomes, or their remodeling activities might be dependent on the nature and/or location of the damage within the nucleosome. Here we show that two different chromatin remodeling activities (SWI/SNF and ISW2) can act on UV-damaged nucleosomes and facilitate repair by photolyase. In addition, the photoreactivation pattern of remodeled nucleosomes reflected differences in the activities of SWI/SNF and ISW2. TheHindIII/BamHI fragment of p8ATDED (31Losa R. Omari S. Thoma F. Nucleic Acids Res. 1990; 18: 3495-3502Crossref PubMed Scopus (68) Google Scholar) was subcloned in either orientation into the SacI site of pUC18 to generate p18ATDED and p18ATDED-c for top and bottom strand labeling, respectively. The 226-bp fragments generated by cleavage withSmaI and EcoRI were purified and the 3′ ends labeled by filling the recessed ends with [α-32P]dATP and dTTP. Reconstitution was done by histone octamer transfer from chicken erythrocytes core particles (20Schieferstein U. Thoma F. EMBO J. 1998; 17: 306-316Crossref PubMed Scopus (45) Google Scholar). 200 ng of end-labeled DNA was incubated with a 40-fold excess of core particles (8 μg) in 200 μl of 10 mm Tris (pH 7.5), 1 mm EDTA (pH 7.5), 0.8 m NaCl at 37 °C for 30 min and at 4 °C for another 30 min. The samples were dialyzed at 4 °C in a microdialyzer (Pierce, membrane molecular weight cut-off 8000) overnight against dialysis buffer 1 (0.6 m NaCl, 10 mm Tris (pH 7.5), 1 mm EDTA (pH 7.5), 50 μm phenylmethylsulfonyl fluoride), 4–5 h against dialysis buffer 2 (10 mm Tris (pH 7.5), 50 mmNaCl, 1 mm EDTA (pH 7.5), 50 μmphenylmethylsulfonyl fluoride), and finally 2–3 h against fresh dialysis buffer 2. Yeast SWI/SNF was added to nucleosomes (160 ng for the DNase I footprint, 100 ng for the UV footprint, and 800 ng for the photoreactivation experiments), and the buffer was adjusted to final concentrations of 8 mmTris (pH 7.5), 100 mm NaCl, 1 mmdithiothreitol, 5 mm MgCl2, 3% glycerol (in final volumes of 30, 20, and 80 μl, respectively). Where indicated, ATP was added to a final concentration of 0.5 or 1 mm. The photoreactivation experiment contained in addition 46 μg/ml insulin brought in by a new yeast SWI/SNF batch. The molar ratios of ySWI/SNF to nucleosomes were 1 (10 ng of complex/ng of nucleosome) for the DNase I footprinting (Fig. 3, A and B), 0.8 (8 ng of complex/ng of nucleosome) for the UV photo-footprinting (Fig. 3,B and C), and 0.6 (6 ng of complex/ng of nucleosome) for the photoreactivation experiments (Fig. 4). The reactions were incubated 30 min at 30 °C. For competition, 2-μl aliquots were removed, and an excess of linear plasmid DNA (pBSFT99; 1 μg in 10 mm Tris (pH 7.5), 100 mm NaCl, 3% glycerol) was added, resulting in a final volume of 5 μl. The reaction was incubated for 45 min at room temperature.Figure 4Enhanced CPD repair on nucleosomes remodeled by ySWI/SNF. DNA was end-labeled on the bottom strand, reconstituted in nucleosomes, irradiated with 500 J/m2 UV, incubated with ySWI/SNF (0.6 complex per nucleosome), and exposed to photolyase and photoreactivating light for up to 60 min. A, nucleoprotein gel. D, naked DNA; N andNucl., nucleosomes; W, wells. B, nucleoprotein gel analysis after competition of ySWI/SNF. The samples described in A were incubated with an excess plasmid DNA for 45 min at room temperature. The fraction of material in the well (W), the nucleosomal bands (N), and the naked DNA bands (D) is indicated for each lane (bottom).C, photoreactivation of naked DNA, nucleosomes, and remodeled nucleosomes. Description is as in Fig. 2B. Lane 2, initial CPD distribution; lanes 4–6, photoreactivation in nucleosomes; lanes 7–9, photoreactivation of nucleosomes and ySWI/SNF; lanes 10–12, photoreactivation of nucleosomes, ySWI/SNF and ATP; lanes 13–15, photoreactivation of naked DNA isolated from irradiated nucleosomes; lanes 1 and 3, damaged DNA treated with no T4-endoV and an excess of T4-endoV, respectively;ellipse, the position of the histone octamer; black bars 10–19, pyrimidine clusters. D, fraction of CPD repaired in 30 min in clusters 10–19. Nucleosomes (open bars), nucleosomes incubated with ySWI/SNF (gray bars), nucleosomes incubated with SWI/SNF and ATP (black bars). (Data of Figs. 3 and 4 are from three independent experiments reproducing ATP-dependent modulation of DNA accessibility (Fig. 3B and Fig. 4, C andD) and DNA structure (Fig. 3D and Fig.4C).)View Large Image Figure ViewerDownload (PPT) The yISW2 complex was expressed using the Bac to Bac baculovirus expression system (Invitrogen). SF21 cells were grown in suspension culture to a density of 1 × 106 cells/ml in SF900 media and infected with viral m.o.i. of 0.1 or 1.0 using a C-terminally His6-tagged version of ISW2. Cells were harvested by centrifugation 48 (for 1.0 m.o.i.) or 72 h (for 0.1 m.o.i.) post-infection. All subsequent steps were performed at 4 °C. Cell lysis and binding to Talon metal affinity resin was carried out according to the manufacturer's instructions (Clontech) except that the binding buffer was adjusted to pH 7.4. Elution was carried out with a linear gradient to 100 mm imidazole. Eluted material was loaded directly onto a Porous HPQ column (Beckman Instruments) and eluted with a linear gradient to 0.6 m KCl. Combined fractions were loaded on a Sepharose G-400 gel filtration column run in 100 mm KCl, 20 mm Tris (pH 7.5). Peak fractions were concentrated using Vivaspin concentrators (Sartorius). His-tagged yISW2 was added to nucleosomes (2 μg), and the buffer was adjusted to final concentrations of 10 mm Tris (pH 7.5), 90 mm NaCl, 5 mmMgCl2, 1 mm dithiothreitol, 8% glycerol, 30 μg/ml bovine serum albumin (in a final volume of 100 μl). Where indicated, ATP was added to a final concentration of 0.5 mm. The molar ratio of ISW2 to nucleosomes was 0.7 (1 ng of complex/ng of nucleosome). The reactions were incubated for 30 min at 30 °C. Nucleosome (40 ng/μl), remodeled nucleosome (5 ng/μl), and DNA (5–40 ng/μl) samples were split in 20–40-μl droplets and irradiated on parafilm strips placed on ice at a fluence of 15 watts/m2 using germicidal lamps (G15WT8, Sylvania) emitting predominantly at 254 nm. Irradiated nucleosomes or naked DNA isolated from irradiated nucleosomes were mixed with Escherichia coli photolyase (BD Biosciences) to yield a ratio of 70–75 ng of photolyase/μg of DNA. Photoreactivation was performed by irradiating the samples at 30 °C with six fluorescent lamps (15 watts, F15T8 BLB, Sylvania, peak emission at 375 nm) with a fluence of 17 watts/m2. In the SWI/SNF and ISW2 remodeling experiments, samples were photoreactivated in remodeling buffer. SDS was added to the repair samples (0.5% final concentration), followed by proteinase K digestion. DNA was purified through QIAquick columns (Qiagen) and eluted in 50 mm Tris, 5 mm EDTA. DNA was incubated at 37 °C; T4-endonuclease V (Epicentre) was added (0.06 units/ng DNA), and incubation was continued for 3 h. DNA was purified by StrataClean resin (Stratagene) extraction, precipitated, and analyzed on denaturing 8% acrylamide gels. Reconstitution products were analyzed on 4% acrylamide gels run in 0.5× TBE. Where indicated, the nucleoprotein gels also contained 10% glycerol (see figure legends). Prior to loading, the samples were mixed with glycerol to yield a final concentration of 6% glycerol and electrophoresed at 4 °C for 3–4 h at 12 mA. DNase I digestion of nucleosomal and naked DNA samples was performed in parallel. The samples were adjusted to 5 mm MgCl2 and incubated with DNase I (Roche Applied Science; 2 units of DNase I/μg of nucleosomal DNA and 0.2 units of DNase I/μg of naked DNA) at room temperature between 30 s and 6 min. The reactions were stopped by adjusting the samples to 10 mm EDTA. The DNA was purified and analyzed on 8% denaturing acrylamide gels. 1.8 μg of nucleosomes or naked DNA was incubated with 50 units of restriction enzyme (AflIII, HhaI, and XhoI) at 30 °C for 3 h in a buffer adjusted to 100 mm NaCl, 10 mm MgCl2, 1 mm dithiothreitol, 50 mm Tris (pH 7.5), and 0.1 mg/ml bovine serum albumin for the restriction enzymes AflIII and HhaI. Purified DNA was analyzed on 8% denaturing acrylamide gels. The gels were dried on Whatman DE81 and 3MM papers and quantified using a PhosphorImager (Amersham Biosciences). We reconstituted a nucleosome at the end of a 226-bp DNA fragment. This “ATDED-long” fragment originates from the natural yeast DED1 promoter and contains several polypyrimidine tracts (31Losa R. Omari S. Thoma F. Nucleic Acids Res. 1990; 18: 3495-3502Crossref PubMed Scopus (68) Google Scholar), which allowed us to monitor DNA structure by UV footprinting and DNA damage accessibility by photolyase (Fig.1A). This nucleosome has the space to reassemble or slide to alternative positions as a consequence of DNA damage formation and interactions with DNA repair enzymes and remodeling complexes. The DNA was end-labeled with 32P on either strand separately, and nucleosomes were reconstituted by histone octamer transfer from chicken erythrocytes core particles. Nucleoprotein gels showed that reconstitution was efficient, with over 90% of the DNA folded into nucleosomes. Only one preferential product was observed (see examples below). DNase I digestion produced a 10-bp repeat pattern between map units 1360 and 1497, which is characteristic for nucleosomes occupying a single rotational orientation (Fig. 1B, lanes 2 and 3; Fig. 3B, lanes 5and 6). No such patterns were generated in naked DNA (Fig. 1B, lanes 4). The translational position of the nucleosome was verified by digestion with restriction enzymes. Reconstituted DNA was efficiently cleaved by AflIII (84%) and HhaI (79%), whereas cleavage by XhoI was strongly inhibited (2% cut; Fig. 1C). Thus, DNase I and restriction analyses demonstrate that the nucleosome is positioned at the right end of the DNA and adopts a preferential rotational setting. For repair experiments, reconstituted nucleosomes were irradiated with UV light at a dose of 750 J/m2 to generate about 1.5 CPDs per fragment and exposed to E. coli DNA photolyase and photoreactivating light for up to 120 min at 30 °C. Nucleoprotein gels revealed that the nucleosomal fraction remained unchanged after UV irradiation and during photoreactivation (Fig.2A, lanes 3–9). Hence, neither UV irradiation nor photolyase disrupted the nucleosome. To assess the CPD distribution and their removal by photolyase, DNA of UV-irradiated and photoreactivated nucleosomes was purified and cut at CPDs with T4-endonuclease V, and the digestion products were displayed by gel electrophoresis (Fig. 2B, lanes 2–9). To test the activity of photolyase on naked DNA, DNA of UV-irradiated nucleosomes was extracted and exposed to photolyase and light for up to 45 min (lanes 10–13). Decreasing band intensities with increasing photoreactivation times showed site-specific repair. Damages were quantified in pyrimidine clusters; the fraction of CPDs repaired in 30 min is shown (Fig. 2C). CPD repair in naked DNA was fast with 65–95% of the CPDs removed in 30 min. In reconstituted samples, repair was fast outside of the predicted nucleosome (clusters 1, 2, 3, 10, and 11) but slow and inefficient in the nucleosome (clusters 5–9 and 13–19). Clusters 12 and 4 are located toward the left end of the nucleosome (on the “linker side”) and were repaired more efficiently than those located in the central region and toward the other end of the nucleosome. Interestingly, cluster 12 (map units 1376–1378) appears to coincide with a DNase I-sensitive site that is not part of the 10-bp ladder and therefore might indicate an unusual structure at the end of the nucleosome (Fig. 1B, bottom strand). In conclusion, CPDs in nucleosomes are resistant to photoreactivation, whereas CPDs outside are efficiently repaired. The results imply that the nucleosome was not displaced either by UV damage formation or by incubation with photolyase despite the length of the fragment. Thus, UV lesions and E. coli photolyase are unable to displace the histone octamer. In contrast to the results obtained in vitro, DNA lesions are completely removed from nucleosomesin vivo. Therefore, we tested whether nucleosome remodeling activities can act on UV-damaged nucleosomes and facilitate CPD accessibility and repair. We first tested ySWI/SNF. SWI/SNF is known to generally enhance accessibility of nucleosomes to DNase I, restriction endonucleases, and transcription factors in vitro, without disruption of the histone octamer (32Cote J. Quinn J. Workman J.L. Peterson C.L. Science. 1994; 265: 53-60Crossref PubMed Scopus (724) Google Scholar, 33Logie C. Peterson C.L. EMBO J. 1997; 16: 6772-6782Crossref PubMed Scopus (167) Google Scholar, 34Owen-Hughes T. Utley R.T. Cote J. Peterson C.L. Workman J.L. Science. 1996; 273: 513-516Crossref PubMed Scopus (197) Google Scholar, 35Utley R.T. Cote J. Owen-Hughes T. Workman J.L. J. Biol. Chem. 1997; 272: 12642-12649Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Reconstituted nucleosomes were incubated with ySWI/SNF either in the absence or in the presence of ATP. In the nucleoprotein gel, all labeled DNA was found in the well after addition of ySWI/SNF (Fig.3A, lanes 3 and 4) and ATP (lane 4), indicating that the nucleosomes were bound to the remodeling complex. DNase I footprinting revealed that the rotational setting of the nucleosome was maintained when complexed with ySWI/SNF in the absence of ATP and lost after incubation with ySWI/SNF and ATP (Fig. 3B, lanes 5–10). The resulting cutting pattern was similar to naked DNA (lanes 2–4). Thus the complex apparently remodels the ATDED-long nucleosome as described for other substrates (32Cote J. Quinn J. Workman J.L. Peterson C.L. Science. 1994; 265: 53-60Crossref PubMed Scopus (724) Google Scholar, 34Owen-Hughes T. Utley R.T. Cote J. Peterson C.L. Workman J.L. Science. 1996; 273: 513-516Crossref PubMed Scopus (197) Google Scholar, 36Cote J. Peterson C.L. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4947-4952Crossref PubMed Scopus (166) Google Scholar). CPD formation depends on the structure of DNA and can be altered by folding of DNA in nucleosomes (37Gale J.M. Smerdon M.J. J. Mol. Biol. 1988; 204: 949-958Crossref PubMed Scopus (63) Google Scholar, 38Schieferstein U. Thoma F. Biochemistry. 1996; 35: 7705-7714Crossref PubMed Scopus (48) Google Scholar, 39Liu X. Mann D.B. Suquet C. Springer D.L. Smerdon M.J. Biochemistry. 2000; 39: 557-566Crossref PubMed Scopus (24) Google Scholar). ATDED-long nucleosomes were analyzed by UV photo-footprinting during remodeling (Fig. 3,C and D). Naked DNA, nucleosomes, and nucleosomes treated with ySWI/SNF in the presence or absence of ATP were irradiated with UV light at a dose of 500 J/m2 to generate about one CPD per fragment. The nucleoprotein gel confirmed that prior to irradiation, all nucleosomes were complexed with ySWI/SNF in the presence and absence of ATP (Fig. 3C, lanes 3 and4). In naked DNA, the CPD formation pattern was heterogeneous, depending on the sequence and the unusual structure of T-tracts which forms characteristic damage patterns (38Schieferstein U. Thoma F. Biochemistry. 1996; 35: 7705-7714Crossref PubMed Scopus (48) Google Scholar, 40Lyamichev V. Nucleic Acids Res. 1991; 19: 4491-4496Crossref PubMed Scopus (41) Google Scholar) (Fig.3D, lane 4, clusters 13 and 16). The damage pattern generated in reconstituted nucleosomes was clearly different (Fig. 3D, lane 5) demonstrating that folding of ATDED-DNA in a nucleosome alters its DNA structure. In particular, folding of DNA into nucleosomes induced enhanced CPD formation in cluster 16 and at the 5′ end of cluster 13. The nucleosome pattern was maintained when nucleosomes were irradiated in the presence of ySWI/SNF (Fig. 3D, lane 6), but irradiation in the presence of ySWI/SNF and ATP generated a pattern similar to that of naked DNA (lane 7). These UV photo-footprinting results demonstrate a change in the structure upon folding of DNA into nucleosomes and upon remodeling by ySWI/SNF. To test whether DNA might have been released from nucleosomes by ySWI/SNF under our conditions, aliquots were incubated with an excess of plasmid DNA to compete for ySWI/SNF and analyzed by nucleoprotein gel electrophoresis (Fig. 3C, lanes 5–8). After competition with plasmid DNA, only a small fraction of material was observed as free DNA, whereas most of the material was found in nucleosomal fractions (Fig. 3C, lanes 7 and 8). This is in agreement with previous work (34Owen-Hughes T. Utley R.T. Cote J. Peterson C.L. Workman J.L. Science. 1996; 273: 513-516Crossref PubMed Scopus (197) Google Scholar, 36Cote J. Peterson C.L. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4947-4952Crossref PubMed Scopus (166) Google Scholar, 41Guyon J.R. Narlikar G.J. Sif S. Kingston R.E. Mol. Cell. Biol. 1999; 19: 2088-2097Crossref PubMed Scopus (56) Google Scholar, 42Sengupta S.M. VanKanegan M. Persinger J. Logie C. Cairns B.R. Peterson C.L. Bartholomew B. J. Biol. Chem. 2001; 276: 12636-12644Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), where no or very little increase in naked DNA was observed in similar competition experiments. Two major nucleosomal bands (Fig. 3C, N and N′) indicate that some nucleosomes might have joined to form dimers following interactions with ySWI/SNF. In summary, the nucleoprotein gel demonstrates that ySWI/SNF did not promote a release of free DNA from nucleosomes. Thus, the UV damage pattern observed by footprinting reflects the pattern of a nucleosome interacting with ySWI/SNF or being remodeled by ySWI/SNF. To address a possible contribution of nucleosome remodeling activities to DNA repair, nucleosomes were irradiated with UV light at a dose of 500 J/m2, incubated with ySWI/SNF either in the absence or presence of ATP, and exposed to photolyase in the presence of photoreactivating light. The nucleoprotein gel (Fig.4A) showed that the nucleosomes were stable over t
Graphical Abstract Abstract figure legend HCN channels play an evolutionarily conserved pacemaker role in renal pelvic smooth muscle (RPSM) of lower and higher order mammals. The function of hyperpolarization-activated cation (HCN) channels in smooth muscle pacemakers remains controversial. Renal pelvic smooth muscle pacemakers trigger smooth muscle contractions that expel waste from the kidney, and HCN channels have been localized to these pacemaker tissues. To date, however, the mechanisms underlying RPSM pacemaker activity remain elusive. RPSM pacemaker activity was investigated in both lower (top left) and higher order (bottom left) mammalian models, which exhibit divergent upper urinary tract anatomies. We performed morphological and functional studies from the single-molecule to the whole-organ level and showed that HCN channels drive RPSM pacemaker activity. RPSM pacemakers (boxed regions) integrated into the muscular syncytium, expressed HCN channels on their plasmalemma and exhibited the Ih ‘funny’ pacemaker current conducted by HCN channels. Critically, HCN channel block abolished electrical pacemaker activity and peristaltic smooth muscle contractions in both lower and higher order mammalian upper urinary tracts. Thus, HCN channels play an evolutionarily conserved pacemaker role in RPSM pacemakers.
We studied endocytosis in chromaffin cells with both perforated patch and whole cell configurations of the patch clamp technique using cell capacitance measurements in combination with amperometric catecholamine detection. We found that chromaffin cells exhibit two relatively rapid, kinetically distinct forms of stimulus-coupled endocytosis. A more prevalent “compensatory” retrieval occurs reproducibly after stimulation, recovering an approximately equivalent amount of membrane as added through the immediately preceding exocytosis. Membrane is retrieved through compensatory endocytosis at an initial rate of ∼6 fF/s. Compensatory endocytotic activity vanishes within a few minutes in the whole cell configuration. A second form of triggered membrane retrieval, termed “excess” retrieval, occurs only above a certain stimulus threshold and proceeds at a faster initial rate of ∼248 fF/s. It typically undershoots the capacitance value preceding the stimulus, and its magnitude has no clear relationship to the amount of membrane added through the immediately preceding exocytotic event. Excess endocytotic activity persists in the whole cell configuration. Thus, two kinetically distinct forms of endocytosis coexist in intact cells during perforated patch recording. Both are fast enough to retrieve membrane after exocytosis within a few seconds. We argue that the slower one, termed compensatory endocytosis, exhibits properties that make it the most likely mechanism for membrane recycling during normal secretory activity.
