RESUMEN
Resolution of Holliday junctions into separate DNA duplexes requires enzymatic cleavage of an equivalent strand from each contributing duplex at or close to the point of strand exchange. Diverse Holliday junction-resolving enzymes have been identified in bacteria, bacteriophages, archaea and pox viruses, but the only eukaryotic examples identified so far are those from fungal mitochondria. We have now determined the crystal structure of Ydc2 (also known as SpCce1), a Holliday junction resolvase from the fission yeast Schizosaccharomyces pombe that is involved in the maintenance of mitochondrial DNA. This first structure of a eukaryotic Holliday junction resolvase confirms a distant evolutionary relationship to the bacterial RuvC family, but reveals structural features which are unique to the eukaryotic enzymes. Detailed analysis of the dimeric structure suggests mechanisms for junction isomerization and communication between the two active sites, and together with site-directed mutagenesis identifies residues involved in catalysis.
Asunto(s)
Cristalografía por Rayos X , Endodesoxirribonucleasas/química , Mitocondrias/enzimología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/enzimología , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Dominio Catalítico , Clonación Molecular , Dimerización , Evolución Molecular , Isomerismo , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Sistemas de Lectura Abierta , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
The Mycobacterium leprae RuvA homologue (MlRuvA) was over-expressed in Escherichia coli and purified to homogeneity. The DNA-binding specificity and the functional interactions of MlRuvA with E. coli RuvB and RuvC (EcRuvB and EcRuvC) were examined using synthetic Holliday junctions. MlRuvA bound specifically to Holliday junctions and produced similar band-shift patterns as EcRuvA. Moreover, MlRuvA formed functional DNA helicase and branch-migration enzymes with EcRuvB, although the heterologous enzyme had a lower efficiency. These results demonstrate that the RuvA homologue of M. leprae is a functional branch-migration subunit. Whereas MlRuvA promoted branch-migration in combination with EcRuvB, it was unable to stimulate branch-migration-dependent resolution in a RuvABC complex. The inability to stimulate RuvC was not due to its failure to form heterologous RuvABC complexes on junctions, since such complexes were detected by co-immunoprecipitation. Most likely, the stability of the heterologous RuvABC complex and, possibly, the interactions between RuvA and RuvC were impaired, as gel-shift experiments failed to show mixed MlRuvA-EcRuvC-junction complexes. These results demonstrate that branch-migration per se and the assembly of a RuvABC complex on the Holliday junction are insufficient for RuvAB-dependent resolution of the junction by RuvC, suggesting that specific and intimate interactions between all three proteins are required for the function of a RuvABC "resolvasome".
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Mycobacterium leprae/enzimología , Recombinación Genética , Adenosina Trifosfatasas/metabolismo , Calcio/metabolismo , Calcio/farmacología , Cationes Bivalentes/metabolismo , Cationes Bivalentes/farmacología , ADN/química , ADN/genética , ADN/metabolismo , ADN Helicasas/metabolismo , Escherichia coli/genética , Sustancias Macromoleculares , Magnesio/metabolismo , Magnesio/farmacología , Mycobacterium leprae/genética , Unión Proteica/efectos de los fármacos , Recombinación Genética/genética , Especificidad por SustratoRESUMEN
The interaction between homologous DNA molecules in recombination and DNA repair leads to the formation of crossover intermediates known as Holliday junctions. Their enzymatic processing by the RuvABC system in bacteria involves the formation of a complex between RuvA and the Holliday junction. To study the solution structure of this complex, contrast variation by neutron scattering was applied to Mycobacterium leprae RuvA (MleRuvA), a synthetic analogue of a Holliday junction with 16 base-pairs in each arm, and their stable complex. Unbound MleRuvA was octameric in solution, and formed an octameric complex with the DNA junction. The radii of gyration at infinite contrast were determined to be 3.65 nm, 2.74 nm and 4.15 nm for MleRuvA, DNA junction and their complex, respectively, showing that the complex was structurally more extended than MleRuvA. No difference was observed in the presence or absence of Mg2+. The large difference in RG values for the free and complexed protein in 65% 2H2O, where the DNA component is "invisible", showed that a substantial structural change had occurred in complexed MleRuvA. The slopes of the Stuhrmann plots for MleRuvA and the complex were 19 and 15 or less (x10(-5)), respectively, indicating that DNA passed through the centre of the complex. Automated constrained molecular modelling based on the Escherichia coli RuvA crystal structure demonstrated that the scattering curve of octameric MleRuvA in 65% and 100% 2H2O is explained by a face-to-face association of two MleRuvA tetramers stabilised by salt-bridges. The corresponding modelling of the complex in 65% 2H2O showed that the two tetramers are separated by a void space of about 1-2 nm, which can accommodate the width of B-form DNA. Minor conformational changes between unbound and complexed MleRuvA may occur. These observations show that RuvA plays a more complex role in homologous recombination than previously thought.
Asunto(s)
ADN Helicasas , Proteínas de Unión al ADN/química , ADN/química , Mycobacterium leprae/genética , Oligodesoxirribonucleótidos/química , Recombinación Genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Simulación por Computador , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , Neutrones , Oligodesoxirribonucleótidos/metabolismo , Unión Proteica , Dispersión de Radiación , SolucionesRESUMEN
Holliday junctions occur as intermediates in homologous recombination and DNA repair. In bacteria, resolution of Holliday junctions is accomplished by the RuvABC system, consisting of a junction-specific helicase complex RuvAB, which promotes branch migration, and a junction-specific endonuclease RuvC, which nicks two strands. The crystal structure of a complex between the RuvA protein of M. leprae and a synthetic four-way junction has now been determined. Rather than binding on the open surface of a RuvA tetramer as previously suggested, the DNA is sandwiched between two RuvA tetramers, which form a closed octameric shell, stabilized by a conserved tetramer-tetramer interface. Interactions between the DNA backbone and helix-hairpin-helix motifs from both tetramers suggest a mechanism for strand separation promoted by RuvA.
Asunto(s)
ADN Helicasas , ADN Bacteriano/química , Proteínas de Unión al ADN/química , Conformación Proteica , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Sitios de Unión , Cristalografía por Rayos X/métodos , Escherichia coli , Proteínas de Escherichia coli , Sustancias Macromoleculares , Modelos Moleculares , Mycobacterium leprae , Pliegue de Proteína , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
BACKGROUND: The Escherichia coli RuvA and RuvB proteins promote the branch migration of 4-way (Holliday) junctions during genetic recombination. The active complex is a tripartite structure in which RuvA protein is bound to the crossover and is sandwiched between two hexameric rings of RuvB. Branch migration requires ATP hydrolysis and occurs as the DNA passes through each RuvB ring. RESULTS: In this work, we have investigated the mechanism by which RuvAB catalyses the branch migration of a three-armed (Y) junction. Using synthetic DNA structures, we observed the formation of DNA products, a partial duplex DNA molecule and a single-stranded oligonucleotide, indicative of a branch migration reaction that occurred with unique polarity. Analysis of the RuvAB-junction complex by DNase footprinting showed that RuvA bound asymmetrically to the junction and targeted a single hexameric RuvB ring to one arm of DNA. CONCLUSION: Branch migration of a three-armed junction occurs in a unidirectional manner that is determined by the assembly of a single RuvB ring onto one arm of the DNA. The asymmetry of the complex and observed directionality of branch migration indicate that strand passage occurs as the DNA is pulled into the RuvB ring structure, a reaction likely to be coupled with DNA unwinding.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , ADN Helicasas , ADN Bacteriano/química , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Secuencia de Bases , Sitios de Unión , Huella de ADN , ADN Bacteriano/genética , Desoxirribonucleasa I , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Conformación de Ácido Nucleico , Conformación Proteica , Recombinación GenéticaRESUMEN
The RuvA and RuvB proteins of Escherichia coli act late in recombination and DNA repair to catalyze the branch migration of Holliday junctions made by RecA. In this paper, we show that addition of RuvAB to supercoiled DNA that is bound by RecA leads to the rapid dissociation of the RecA nucleoprotein filament, as determined by a topological assay that measures DNA underwinding and a restriction endonuclease protection assay. Disruption of the RecA filament requires RuvA, RuvB, and hydrolysis of ATP. These findings suggest several important roles for the RuvAB helicase during genetic recombination and DNA repair: (i) displacement of RecA filaments from double-stranded DNA, (ii) interruption of RecA-mediated strand exchange, (iii) RuvAB-catalyzed branch migration, and (iv) recycling of RecA protein.
Asunto(s)
Proteínas Bacterianas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Plásmidos/metabolismo , Rec A Recombinasas/metabolismo , Proteínas Bacterianas/aislamiento & purificación , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo I/aislamiento & purificación , ADN-Topoisomerasas de Tipo I/metabolismo , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Agar , Proteínas de Escherichia coli , Cinética , Modelos Estructurales , Conformación de Ácido Nucleico , Plásmidos/química , Plásmidos/aislamiento & purificación , Rec A Recombinasas/química , Mapeo Restrictivo , Triticum/enzimologíaRESUMEN
The RuvA and RuvB proteins of Escherichia coli promote the branch migration of Holliday junctions in vitro. To understand the relationship between branch migration and the intrinsic 5'-->3' DNA helicase activity of RuvAB, the requirements and substrate specificity of the helicase reaction have been studied in more detail. We find that RuvAB-mediated DNA unwinding and branch migration reactions show similar requirements for Mg2+ and ATP and are inhibited to a similar extent by ADP and ATP gamma S (adenosine 5'-O-(3-thiotriphosphate)). The helicase activity, measured by the dissociation of a short fragment from circular single-stranded DNA, requires both RuvA and RuvB and is stimulated by subsaturating concentrations of single-strand binding protein (SSB). In contrast, saturating concentrations of SSB are inhibitory. Using substrates that contain a DNA junction, which permits the specific binding of RuvA, we find that the RuvA and RuvB proteins promote two types of helicase reactions: nonspecific reactions, which are sensitive to inhibition by stoichiometric amounts of SSB, and junction-targeted reactions, which are not inhibited by SSB. Using three-armed structures, we observe that junction-targeted reactions display a polarity and result in asymmetric product formation. Junction-specific binding and the subsequent initiation of DNA unwinding are likely to represent early steps in the process of branch migration.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Adenosina Trifosfatasas/metabolismo , Secuencia de Bases , ADN/metabolismo , Daño del ADN , Proteínas de Escherichia coli , Datos de Secuencia MolecularRESUMEN
The RuvB protein is induced in Escherichia coli as part of the SOS response to DNA damage. It is required for genetic recombination and the postreplication repair of DNA. In vitro, the RuvB protein promotes the branch migration of Holliday junctions and has a DNA helicase activity in reactions that require ATP hydrolysis. We have used electron microscopy, image analysis, and three-dimensional reconstruction to show that the RuvB protein, in the presence of ATP, forms a dodecamer on double-stranded DNA in which two stacked hexameric rings encircle the DNA and are oriented in opposite directions with D6 symmetry. Although helicases are ubiquitous and essential for many aspects of DNA repair, replication, and transcription, three-dimensional reconstruction of a helicase has not yet been reported, to our knowledge. The structural arrangement that is seen may be common to other helicases, such as the simian virus 40 large tumor antigen.
Asunto(s)
Proteínas Bacterianas/ultraestructura , ADN Helicasas/ultraestructura , ADN Bacteriano/ultraestructura , Escherichia coli/ultraestructura , Algoritmos , Procesamiento de Imagen Asistido por Computador , Microscopía Electrónica de Rastreo , Modelos MolecularesRESUMEN
The Escherichia coli RuvA and RuvB proteins mediate the branch migration of Holliday junctions in vitro. In the presence of stoichiometric amounts of RuvB (1 RuvB dimer/12 nucleotides), branch migration can occur without need for RuvA. However, RuvA is required when the RuvB concentration is reduced 4-fold or more. Under optimal conditions, we found the minimal protein requirement to be 1 RuvB dimer per 500-1100 nucleotides and 1 RuvA tetramer per 600-1200 nucleotides. To determine the roles of RuvA and RuvB in branch migration, we compared branch migration reactions mediated by RuvB only and by RuvA and RuvB. The time courses of the two reactions were similar, and both required ATP and Mg2+. However, RuvB-mediated branch migration occurred at lower ATP concentrations (> or = 200 microM) and higher Mg2+ concentrations (> or = 10 mM MgCl2) than the reaction mediated by RuvA and RuvB (> or = 1 mM ATP, > or = 5 mM MgCl2). The Mg2+ requirement for RuvB-mediated branch migration reflects the Mg2+ requirement of RuvB for DNA binding (Müller, B., Tsaneva, I.R., and West, S. C. (1993) J. Biol. Chem. 268, 17185-17189) and can be overcome by addition of RuvA. These results indicate that RuvA protein facilitates the interaction of RuvB with DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli , CinéticaRESUMEN
Using recombination intermediates made by RecA protein, we have shown that the Escherichia coli RuvB protein can mediate the branch migration of Holliday junctions in vitro. The reaction is dependent on the presence of > or = 10 mM Mg2+ and stoichiometric amounts of RuvB. The presence of E. coli RuvA protein reduces the requirement for Mg2+ and also the stoichiometric requirement for RuvB (Müller, B., Tsaneva, I. R., and West, S. C. (1993) J. Biol. Chem. 268, 17179-17184). To determine the roles of the two proteins during branch migration, we have investigated the interaction of RuvB with DNA in the absence or presence of RuvA, by (i) gel retardation of protein-DNA complexes, (ii) stimulation of the RuvB ATPase, and (iii) protection of DNA from DNase I. The interaction of RuvB with duplex DNA was Mg(2+)-dependent and correlated with the Mg2+ requirement of the RuvB-mediated branch migration reaction. RuvB also interacted with ssDNA, but the affinity was significantly lower than for duplex DNA. In contrast to RuvB, the interaction of RuvA with duplex DNA occurred in the absence of Mg2+ and was inhibited by Mg2+ in a concentration-dependent manner. At 5 mM Mg2+, RuvA protein facilitated the interaction of RuvB with DNA, leading to the formation of a complex containing RuvA, RuvB, and duplex DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , ADN Bacteriano/metabolismo , Desoxirribonucleasa I/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coliRESUMEN
The SOS-inducible ruvA and ruvB gene products of Escherichia coli are required for normal levels of genetic recombination and DNA repair. In vitro, RuvA protein interacts specifically with Holliday junctions and, together with RuvB (an ATPase), promotes their movement along DNA. This process, known as branch migration, is important for the formation of heteroduplex DNA. In this paper, we show that the RuvA and RuvB proteins promote the unwinding of partially duplex DNA. Using single-stranded circular DNA substrates with annealed fragments (52-558 nucleotides in length), we show that RuvA and RuvB promote strand displacement with a 5'-->3' polarity. The reaction is ATP-dependent and its efficiency is inversely related to the length of the duplex DNA. These results show that the ruvA and ruvB genes encode a DNA helicase that specifically recognizes Holliday junctions and promotes branch migration.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Bacteriófago phi X 174/genética , Bacteriófago phi X 174/metabolismo , ADN Helicasas/genética , Reparación del ADN , ADN Circular/metabolismo , ADN de Cadena Simple/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli , Cinética , Datos de Secuencia Molecular , Mapeo Restrictivo , Homología de Secuencia de Aminoácido , Especificidad por SustratoAsunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Recombinación Genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Reparación del ADN , Replicación del ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Escherichia coli/metabolismo , Genes Bacterianos , Cinética , Modelos GenéticosRESUMEN
The RuvA and RuvB proteins of Escherichia coli play important roles in the post-replicational repair of damaged DNA, genetic recombination and cell division. In this paper, we describe the construction of over expression vectors for RuvA and RuvB and detail simple purification schemes for each protein. The purified 22 kDa RuvA polypeptide forms a tetrameric protein (M(r) ca. 100,000) as observed by gel filtration. The tetramer is stabilised by strong disulphide bridges that resist denaturation during SDS-PAGE (in the absence of boiling and beta-mercaptoethanol). In contrast, purified RuvB polypeptides (37 kDa) weakly associate to form a dimeric protein (M(r) ca. 85,000). At low protein concentrations, the RuvB dimer dissociates into monomers. The multimeric forms of each protein may be covalently linked by the bifunctional cross-linking reagent dimethyl suberimidate. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction was found to stimulate the rate of strand exchange leading to the rapid formation of heteroduplex DNA.
Asunto(s)
Proteínas Bacterianas/metabolismo , ADN Helicasas , Reparación del ADN , ADN Bacteriano/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Cromatografía en Gel , Clonación Molecular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Proteínas de Escherichia coli , Cinética , Recombinación GenéticaRESUMEN
The RuvA and RuvB proteins of E. coli, which are induced as part of the cellular response to DNA damage, act together to promote the branch migration of Holliday junctions. Addition of purified RuvA and RuvB to a RecA-mediated recombination reaction stimulates the rate of strand exchange and the formation of hetero-duplex DNA. Stimulation does not occur via interaction with RecA; instead, RuvA and RuvB act directly upon recombination intermediates (Holliday junctions) made by RecA. We show that RuvAB-mediated branch migration requires ATP and can bypass UV-induced DNA lesions. At high RuvB concentrations, the requirement for RuvA is overcome, indicating that the RuvB ATPase provides the motor force for branch migration. RuvA protein provides specificity by binding to the Holliday junction, thereby reducing the requirement for RuvB by 50-fold. The newly discovered biochemical properties of RuvA, RuvB, and RuvC are incorporated into a model for the postreplicational repair of DNA following UV irradiation.
Asunto(s)
Proteínas Bacterianas/farmacología , ADN Helicasas , Proteínas de Unión al ADN/farmacología , ADN/efectos de los fármacos , Adenosina Trifosfato , Daño del ADN , Reparación del ADN , Escherichia coli/genética , Proteínas de Escherichia coli , Modelos Moleculares , Recombinación Genética/efectos de los fármacosRESUMEN
The nfo (endonuclease IV) gene of Escherichia coli is induced by superoxide generators such as paraquat (methyl viologen). An nfo'-lacZ operon fusion was used to isolate extragenic mutations affecting its expression. The mutations also affected the expression of glucose 6-phosphate dehydrogenase, Mn2(+)-superoxide dismutase (sodA), and three lacZ fusions to soi (superoxide-inducible) genes of unknown function. The mutations were located 2 kilobases clockwise of ssb at 92 min on the current linkage map. One set of mutations, in a new gene designated soxR, caused constitutive overexpression of nfo and the other genes. It included insertions or deletions affecting the carboxyl end of a 17-kilodalton polypeptide. In a soxR mutant, the expression of sodA, unlike that of nfo, was also regulated independently by oxygen tension. Two other mutants were isolated in which the target genes were noninducible; they had an increased sensitivity to killing by superoxide-generating compounds. One had a Tn10 insertion in or near soxR; the other had a multigene deletion encompassing soxR. Therefore, the region functions as a positive regulator because it encodes one or more products needed for the induction of nfo. Regulation is likely to be at the level of transcription because the mutations were able to affect the expression of an nfo'-lac operon fusion that contained the ribosome-binding site for lacZ. Some mutant plasmids that failed to suppress (or complement) constitutivity in trans had insertion mutations several hundred nucleotides upstream of soxR in the general region of a gene for a 13-kilodalton protein encoded by the opposite strand, raising the possibility of a second regulatory gene in this region. The result define a new regulon, controlled by soxR, mediating at least part of the global response to superoxide in E. coli.
Asunto(s)
Endodesoxirribonucleasas/genética , Proteínas de Escherichia coli , Escherichia coli/genética , Genes Reguladores , Operón , Superóxidos/metabolismo , Anaerobiosis , Deleción Cromosómica , Elementos Transponibles de ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Endodesoxirribonucleasas/aislamiento & purificación , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Expresión Génica , Genotipo , Plásmidos , Proteínas Recombinantes de Fusión/aislamiento & purificación , Mapeo Restrictivo , Superóxidos/farmacología , Transcripción Genética , beta-Galactosidasa/genética , beta-Galactosidasa/aislamiento & purificaciónRESUMEN
Calf thymus chromatin was digested with trypsin and the structural alterations which occurred were followed by flow linear dichroism. After a sharp initial increase, the amplitude of the positive signal gradually decreased followed by a change of the sign of the dichroism and further increase of the negative signal up to a plateau. These changes of the dichroism were compared to the respective changes in the histone pattern. It was shown that the positive dichroism of chromatin did not depend on the condensation state of chromatin, and that the orientation of the nucleosomes along the chromatin fiber was maintained by the globular domain of H1 and the non-structured parts of core histones.
Asunto(s)
Cromatina/ultraestructura , Histonas/fisiología , Nucleosomas/ultraestructura , Animales , Bovinos , Cinética , Microscopía Electrónica , Conformación Proteica , Espectrofotometría Ultravioleta , Timo/ultraestructura , TripsinaRESUMEN
The influence of thermal denaturation on the nucleosomal structure of histone-H1-depleted chromatin was studied using psoralen-treated nucleoprotein preparations subjected to partial thermal denaturation. DNA was cross-linked with psoralen to ensure its complete renaturation upon cooling. The structure of the preheated nucleoprotein was investigated by thermal denaturation, kinetics of hydrolysis and DNA fragment pattern obtained upon digestion with micrococcal nuclease. The electron micrographs of the partially denatured nucleohistone showed gross changes in the nucleosomal structure which were consistent with a sliding of histone cores along DNA as recently reported by Tsaneva et al. (Tsaneva, I., Dimitrov, S., Pashev, I. and Tsanev, R., FEBS Lett., (1980) 112, 143-146). This interpretation is strongly supported by the following features of the partially denatured material: a, increased rate of degradation of DNA by micrococcal nuclease; b, melting of a part of DNA as a protein-free DNA; and c, shortening of the DNA repeat length upon digestion with micrococcal nuclease. The sliding of the core histones is parallelled by the denaturation of histones, which accounts for the very intensive background in the DNA digestion pattern, the loss of nucleosome morphology at higher temperatures, and the disappearance in the melting profile of the transition at 72 degrees C.