The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks

Repair of chromosome double-strand breaks (DSBs) is central to cell survival and genome integrity. Nonhomologous end joining (NHEJ) is the major cellular repair pathway that eliminates chromosome DSBs. Here we report our genetic screen that identified Rsc8 and Rsc30, subunits of the Saccharomyces cerevisiae chromatin remodeling complex RSC, as novel NHEJ factors. Deletion of RSC30 gene or the C-terminal truncation of RSC8 impairs NHEJ of a chromosome DSB created by HO endonuclease in vivo. rsc30Delta maintains a robust level of homologous recombination and the damage-induced cell cycle checkpoints. By chromatin immunoprecipitation, we show recruitment of RSC to a chromosome DSB with kinetics congruent with its involvement in NHEJ. Recruitment of RSC to a DSB depends on Mre11, Rsc30, and yKu70 proteins. Rsc1p and Rsc2p, two other RSC subunits, physically interact with yKu80p and Mre11p. The interaction of Rsc1p with Mre11p appears to be vital for survival from genotoxic stress. These results suggest that chromatin remodeling by RSC is important for NHEJ.     

Direct photo-induced DNA strand scission by a ruthenium bipyridyl complex

Irradiation of plasmid DNA in the presence of Ru(II)-2, a modified tris(2,2'-bipyridyl)Ru(II) complex, in which two hydroxamic acid groups are attached to one of the three bipyridyl ligands, results in total fragmentation of the DNA. The photo-chemical reaction products were analyzed by gel electrophoresis, which revealed complete fragmentation. Further evidence for the complete degradation of the DNA was obtained by imaging the pre- and post-treated plasmid DNA using atomic force microscopy (AFM). A mechanism for the reaction is proposed. It initially involves the photo-chemical generation of Ru(III) ions and superoxide radicals, as corroborated by absorbance difference spectroscopy and electron paramagnetic resonance (EPR). Consequently, Ru(III) preferentially oxidizes guanine, liberating superoxide radicals that yield OH radicals. The OH radicals were identified by observing the spectral change at 532 nm of a 5'-dAdG substrate forming a colored adduct with thiobarbituric acid. These radicals are associated with the major non-specific damage exerted to DNA.     

Chromatin remodeling: RSC Motors along the DNA

Single-molecule experiments show that the chromatin-remodeling complex RSC, a member of the SNF2 ATPase family, induces formation of a negatively supercoiled DNA loop by active translocation.     

Direct observation of the biphasic conformational change of DNA induced by cationic polymers.

The interaction between T4 DNA and basic polypeptides was observed using fluorescence microscopy. Free DNA molecules exhibited random Brownian motion accompanying the conformational change. With the addition of polycation, such as histone and polyarginine, DNA molecules tended to shrink to become spherical shapes. The persistent lengths and the distributions of long axis lengths of DNA-polyarginine complexes were determined from the video images at various polyarginine concentrations. It is demonstrated that the conformation of DNA changes in a biphasic manner in the presence of polyarginine.     

Direct observation of the assembly of RecA/DNA complexes by atomic force microscopy

The formation of the RecA/DNA nucleofilament on nicked circular double stranded (ds) DNA in the presence of ATPgammaS was studied using the atomic force microscope (AFM) at nanometer resolution. The AFM allowed simultaneous observation of both dsDNA substrate and RecA protein-coated sections such that they are highly distinguishable. Using a time series of images, the complex formation was monitored. AFM imaging provided direct evidence that assembly of the nucleofilaments occurs via a nucleation and growth mechanism. The nucleation step is much slower than the growth phase, as demonstrated by the predominance of naked dsDNA at early and middle time points, followed by the rapid appearance of partially then fully formed complexes. Observation of the formation of nucleation sites without accompanying growth on unnicked dsDNA enabled an estimate of the nucleation rate, of 5 x 10(-5) RecA min(-1) bp(-1). The published model for the analysis of RecA assembly on dsDNA deduces a single kinetic parameter that prevents the separate determination of nucleation rate and growth rate. By directly measuring the nucleation rate with the AFM, this model is employed to determine a growth rate of 202 min(-1). These AFM results provide the first direct evidence of previous results on complex formation obtained only by indirect means.     

Abundance of the RSC nucleosome-remodeling complex is important for the cells to tolerate DNA damage in Saccharomyces cerevisiae

The essential Nps1p/Sth1p is a catalytic subunit of the nucleosome-remodeling complex, RSC, of Saccharomyces cerevisiae that can alter nucleosome structure by using the energy of ATP hydrolysis. Besides the ATPase domain, Nps1p harbors the bromodomain, of which the function(s) have not yet been defined. We have isolated a temperature-sensitive mutant allele of NPS1, nps1-13, which has amino acid substitutions within the bromodomain. This mutation perturbed the interaction between the RSC components and enhanced the sensitivity of the cells to several DNA-damaging treatments at the permissive temperature. Reduced expression of NPS1 also caused DNA damage sensitivity. These results suggest the importance of the Nps1p bromodomain in RSC integrity and a model in which high amounts of RSC would be required for the cells to overcome DNA damage.     

Direct observation of translocation in individual DNA polymerase complexes

Complexes of phi29 DNA polymerase and DNA fluctuate on the millisecond time scale between two ionic current amplitude states when captured atop the α-hemolysin nanopore in an applied field. The lower amplitude state is stabilized by complementary dNTP and thus corresponds to complexes in the post-translocation state. We have demonstrated that in the upper amplitude state, the DNA is displaced by a distance of one nucleotide from the post-translocation state. We propose that the upper amplitude state corresponds to complexes in the pre-translocation state. Force exerted on the template strand biases the complexes toward the pre-translocation state. Based on the results of voltage and dNTP titrations, we concluded through mathematical modeling that complementary dNTP binds only to the post-translocation state, and we estimated the binding affinity. The equilibrium between the two states is influenced by active site-proximal DNA sequences. Consistent with the assignment of the upper amplitude state as the pre-translocation state, a DNA substrate that favors the pre-translocation state in complexes on the nanopore is a superior substrate in bulk phase for pyrophosphorolysis. There is also a correlation between DNA sequences that bias complexes toward the pre-translocation state and the rate of exonucleolysis in bulk phase, suggesting that during DNA synthesis the pathway for transfer of the primer strand from the polymerase to exonuclease active site initiates in the pre-translocation state.     

Direct observation of the enzyme-intermediate complex of 5-enolpyruvylshikimate-3-phosphate synthase by 13C NMR spectroscopy

The interaction of 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, 4,5-dideoxyshikimate 3-phosphate (ddS3P), and [2-13C]-and [3-13C]phosphoenolpyruvate (PEP) has been examined by 13C NMR spectroscopy. Although no resonances due to a dead-end intermediate complex could be detected, an enzyme active site specific formation of pyruvate was observed. The interaction of EPSP synthase with shikimate 3-phosphate (S3P) and [2-13C]- or [3-13C]PEP has been examined by 13C NMR spectroscopy. With [2-13C]PEP, in addition to the resonances due to [2-13C]PEP and [8-13C]EPSP, new resonances appeared at 164.8, 110.9, and 107.2 ppm. The resonance at 164.8 ppm has been assigned to enzyme-bound EPSP. The resonance at 110.9 ppm has been assigned to C-8 of an enzyme-free tetrahedral intermediate of the sort originally proposed by Levin and Sprinson [Levin, J. G., & Sprinson, D. B. (1964) J. Biol. Chem. 239, 1142-1150] and recently independently observed by Anderson et al. [Anderson, K. S., Sikorski, J. A., Benesi, A. J., & Johnson, K. A. (1988) J. Am. Chem. Soc. 110, 6577-6579]. The resonance at 107.2 ppm has been assigned to an enzyme-bound intermediate whose structure is closely related to that of the tetrahedral intermediate. With [3-13C]PEP, new resonances appeared at 88.9, 26.2, 25.5, and 24.5 ppm. The resonance at 88.9 ppm has been assigned to enzyme-bound EPSP. The resonance at 26.2 ppm, which was found to correlate with 1.48 ppm by isotope-edited multiple quantum coherence 1H NMR spectroscopy, has been assigned to the methyl group 4-hydroxy-4-methylketoglutarate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The BAH domain, polybromo and the RSC chromatin remodelling complex

We isolated Rivulus marmoratus mitochondrial DNA by long-polymerase chain reaction with conserved primers, and sequenced it with 36 sets of internal conserved primers, which were designed from the extensive sequence similarities of mitochondrial DNA from several fish species. The R. marmoratus mitochondrial DNA has 17,329 bp with a conserved structural organization compared to those of other fish. Rivulus marmoratus mitochondrial DNA also has two nearly identical control regions. The basic characteristics of the R. marmoratus mitochondrial genome are discussed.     

Direct observation of the reversible unwinding of a single DNA molecule caused by the intercalation of ethidium bromide

Ethidium bromide (EtBr) is the conventional intercalator for visualizing DNA. Previous studies suggested that EtBr lengthens and unwinds double-stranded DNA (dsDNA). However, no one has observed the unwinding of a single dsDNA molecule during intercalation. We developed a simple method to observe the twisting motions of a single dsDNA molecule under an optical microscope. A short dsDNA was attached to a glass surface of a flow chamber at one end and to a doublet bead as a rotation marker at the other end. After the addition and removal of EtBr, the bead revolved in opposite directions that corresponded to the unwinding and rewinding of a dsDNA, respectively. The amount of intercalating EtBr was estimated from the revolutions of the bead. EtBr occupied 57% of base pairs on a single dsDNA at 1 mM of EtBr, indicating that EtBr molecules could bind at contiguous sites to each other. The isotherm of intercalation showed that negative cooperativity existed between adjoining EtBr molecules. The association constant of EtBr and dsDNA (1.9 (±0.1) × 10⁵ M⁻¹) was consistent with that of previous results. Our system is useful to investigate the twisting of a single dsDNA interacting with various chemicals and biomolecules.     

Direct observation of the alkylation products of deoxyguanosine and DNA by fast atom bombardment mass spectrometry.

By the methods of fast atom bombardment (FAB) mass spectrometry, thin-layer chromatography and ultraviolet absorption spectroscopy adducts have been studied which are formed by an antitumour alkylating drug thiotepa both in a model system, containing only deoxyguanosine (dGuo), and in DNA. Analysis of the model reaction mixture (dGuo + thiotepa) by FAB mass spectrometry permitted observation of adducts dGuo thiotepa, 2dGuo thiotepa, and also the products of their further modification in solution, which occurs by hydrolysis of the glycosidic bond and also by opening of the imidazole ring. In the case of DNA FAB mass spectrometry made it possible to characterize adducts of thiotepa with guanosine (Gua) and adenosine (Ade) without their preliminary purification. The site of alkylation of Gua in both dGuo and DNA is N7, and that of Ade in DNA is N3. The application of the results to the study of the molecular mechanism of the antitumour action of thiotepa is discussed.     

Direct interaction between cohesin complex and DNA replication machinery

Structural maintenance of chromosome 1 (Smc1) is a multifunctional protein, which has been implicated in sister chromatid cohesion, DNA recombination and repair, and the activation of cell cycle checkpoints by ionizing radiation, ultraviolet light, and other genotoxic agents. In order to identify the proteins that interact with Smc1, we conducted the Tandem affinity purification (TAP) technique and analyzed the Smc1-interacting proteins via MALDI-TOF mass spectrometry. We identified minichromosome maintenance 7 (Mcm7), an essential component of the pre-replication complex, as a novel Smc1-interacting protein. Co-immunoprecipitation revealed an interaction occurring between Smc1 and Mcm7, both in vitro and in vivo. Using a GST pull-down assay, we determined that Smc1 interacts physically with Mcm7 via its N-terminal and hinge regions, and Mcm7 interacts with Smc1 via its middle region. Interestingly, we also discovered that Smc1 interacts with other DNA replication proteins, including Mcm6, RFC1, and DNA polymerase alpha. These results suggest that a functional link exists between the cohesin complex and DNA replication proteins.     

Multiple Aspects of ATP-Dependent Nucleosome Translocation by RSC and Mi-2 Are Directed by the Underlying DNA Sequence

Background

Chromosome structure, DNA metabolic processes and cell type identity can all be affected by changing the positions of nucleosomes along chromosomal DNA, a reaction that is catalysed by SNF2-type ATP-driven chromatin remodelers. Recently it was suggested that in vivo, more than 50% of the nucleosome positions can be predicted simply by DNA sequence, especially within promoter regions. This seemingly contrasts with remodeler induced nucleosome mobility. The ability of remodeling enzymes to mobilise nucleosomes over short DNA distances is well documented. However, the nucleosome translocation processivity along DNA remains elusive. Furthermore, it is unknown what determines the initial direction of movement and how new nucleosome positions are adopted.

Methodology/Principal Findings

We have used AFM imaging and high resolution PAGE of mononucleosomes on 600 and 2500 bp DNA molecules to analyze ATP-dependent nucleosome repositioning by native and recombinant SNF2-type enzymes. We report that the underlying DNA sequence can control the initial direction of translocation, translocation distance, as well as the new positions adopted by nucleosomes upon enzymatic mobilization. Within a strong nucleosomal positioning sequence both recombinant Drosophila Mi-2 (CHD-type) and native RSC from yeast (SWI/SNF-type) repositioned the nucleosome at 10 bp intervals, which are intrinsic to the positioning sequence. Furthermore, RSC-catalyzed nucleosome translocation was noticeably more efficient when beyond the influence of this sequence. Interestingly, under limiting ATP conditions RSC preferred to position the nucleosome with 20 bp intervals within the positioning sequence, suggesting that native RSC preferentially translocates nucleosomes with 15 to 25 bp DNA steps.

Conclusions/Significance

Nucleosome repositioning thus appears to be influenced by both remodeler intrinsic and DNA sequence specific properties that interplay to define ATPase-catalyzed repositioning. Here we propose a successive three-step framework consisting of initiation, translocation and release steps to describe SNF2-type enzyme mediated nucleosome translocation along DNA. This conceptual framework helps resolve the apparent paradox between the high abundance of ATP-dependent remodelers per nucleus and the relative success of sequence-based predictions of nucleosome positioning in vivo.     

Direct observation of redox-linked histidine protonation changes in the iron-sulfur protein of the cytochrome bc1 complex by ATR-FTIR spectroscopy

The redox-linked protonation chemistry of the iron-sulfur protein (ISP) of the cytochrome bc(1) complex was studied by analysis of the pH dependencies of redox difference spectra using perfusion/electrochemically induced attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The ISP of Rhodobacter capsulatus within the intact cytochrome bc(1) complex was analyzed in a mutant form in which the midpoint potential of cytochrome c(1) was lower than that of the ISP. This was compared to a soluble domain of the ISP from the bovine bc(1) complex. Spectra of in situ bacterial and isolated bovine proteins differ markedly only in part of their amide I regions with the in situ protein having additional pH-dependent component(s). Apart from this, both in situ and isolated proteins exhibited the same pH-dependent IR features in reduced minus oxidized difference spectra. Specifically, at high pH, a strong H/D insensitive negative band appeared at 1447/1450 cm(-)(1), together with a peak at 1310 cm(-)(1), the change of a 1267/1255 cm(-)(1) peak/trough into a simple 1266 cm(-)(1) peak, and a trough at 1107 cm(-)(1). Comparison with spectra of model materials indicates that all four signals arise from an imidazolate to imidazole transition of histidine, hence providing the first direct demonstration that histidine is the redox-linked protonation site of the ISP. The 1450 cm(-)(1) band can be assigned to a ring stretch that is unique to the imidazolate form of histidine. It is relatively sharp, has a high extinction coefficient, and provides a novel marker band for the detection of imidazolate intermediates in enzymatic mechanisms generally.     

Acetylated histone tail peptides induce structural rearrangements in the RSC chromatin remodeling complex

Post-translational acetylation of histone tails is often required for the recruitment of ATP-dependent chromatin remodelers, which in turn mobilize nucleosomes on the chromatin fiber. Here we show that the lower lobe of the ATP-dependent chromatin remodeler RSC exists in a dynamic equilibrium and can be found extended away or retracted against the tripartite upper lobe of the complex. Extension of the lower lobe increases the size of a central cavity that has been proposed to be the nucleosome binding site. We show that the presence of acetylated histone 3 N-terminal tail peptides stabilizes the lower lobe of RSC in the retracted state, suggesting that domains recognizing the acetylated histone tails reside at the interface between the two lobes. Based on three-dimensional reconstructions, we propose a model for the interaction of RSC with acetylated nucleosomes.