DNA Strand Arrangement within the SfiI−DNA Complex:  Atomic Force Microscopy Analysis

Lushnikov, Alexander Y.; Potaman, Vladimir N.; Oussatcheva, Elena A.; Sinden, Richard R.; Lyubchenko, Yuri L.

doi:10.1021/bi051767c

Cited by 41 publications

(66 citation statements)

References 18 publications

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“…This factor, 1.92, was obtained by dividing the average volume calculated for the SfiI-DNA complexes analyzed by AFM (238 nm 3 ) by the expected molecular mass of SfiI tetramer (124 kDa). Crystallographic analysis of the Sfi-DNA complex (15) has recently corroborated the data obtained using AFM (12). The lengths of 12RSS and 23RSS fragments were used for assigning the fragments in the RAG-RSS complexes.…”

Section: Methodsmentioning

confidence: 64%

“…To estimate the mass of the RAG proteins in the RAG-RSS complexes, a volume-to-mass conversion factor was derived from AFM studies of the SfiI tetramer bound to DNA (12). This factor, 1.92, was obtained by dividing the average volume calculated for the SfiI-DNA complexes analyzed by AFM (238 nm 3 ) by the expected molecular mass of SfiI tetramer (124 kDa).…”

Section: Methodsmentioning

confidence: 99%

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Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Shlyakhtenko

Gilmore

Kriatchko

et al. 2009

Journal of Biological Chemistry

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Two lymphoid cell-specific proteins, RAG1 and RAG2 (RAG), initiate V(D)J recombination by assembling a synaptic complex with recombination signal sequences (RSSs) abutting two different antigen receptor gene coding segments, and then introducing a DNA double strand break at the end of each RSS. Despite the biological importance of this system, the structure of the synaptic complex, and the RAG protein stoichiometry and arrangement of DNA within the synaptosome, remains poorly understood. Here we applied atomic force microscopy to directly visualize and characterize RAG synaptic complexes. We report that the pre-cleavage RAG synaptic complex contains about twice the protein content as a RAG complex bound to a single RSS, with a calculated mass consistent with a pair of RAG heterotetramers. In the synaptic complex, the RSSs are predominantly oriented in a sideby-side configuration with no DNA strand crossover. The mass of the synaptic complex, and the conditions under which it is formed in vitro, favors an association model of assembly in which isolated RAG-RSS complexes undergo synapsis mediated by RAG protein-protein interactions. The replacement of Mg 2؉ cations with Ca 2؉ leads to a dramatic change in protein stoichiometry for all RAG-RSS complexes, suggesting that the cation composition profoundly influences the type of complex assembled.To generate diverse surface antigen receptor molecules, developing lymphocytes undergo a series of site-specific DNA rearrangements to assemble functional antigen receptor genes from component gene segments (1). This DNA rearrangement process, known as V(D)J recombination, is initiated when two lymphoid cell-specific proteins, called RAG1 and RAG2, assemble a multiprotein synaptic complex with a pair of antigen receptor gene segments and subsequently introduce a DNA double strand break at the end of each gene segment (2). A recombination signal sequence (RSS) 3 that abuts each participating gene segment serves as the binding site of the RAG proteins and directs the location of DNA cleavage. Each RSS contains conserved heptamer and nonamer sequences that are separated by either 12 or 23 bp of DNA of more varied sequence (12RSS and 23RSS, respectively); efficient V(D)J recombination generally only occurs between two RSSs in which the lengths of DNA separating the heptamer and nonamer differ (the 12/23 rule). The RAG proteins mediate DNA cleavage via a nick-hairpin mechanism, breaking the DNA between the RSS heptamer and the coding segment; these reaction products are subsequently processed and repaired by the non-homologous end-joining pathway (1, 3).Previous studies suggest that RAG synaptic complexes are assembled through the stepwise binding of a 12RSS followed by the capture of a 23RSS (4 -6). In vitro biochemical studies suggest synapsis is mediated by a RAG1/2 heterotetramer, but there remains disagreement over the stoichiometry of RAG1 in these complexes (7). In addition, fluorescence resonance energy transfer techniques have recently been applied to examine the orientat...

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Section: Methodsmentioning

confidence: 64%

Section: Methodsmentioning

confidence: 99%

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Shlyakhtenko

Gilmore

Kriatchko

et al. 2009

Journal of Biological Chemistry

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“…1a to c). For AFM images, it is possible to estimate the volume of a particle and derive the approximate mass (41,40,42,58,59). The volumes of these foci show a bimodal distribution (Fig.…”

Section: Resultsmentioning

confidence: 99%

MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Ghosh

Horowitz-Scherer

Nikitina

et al. 2010

Molecular and Cellular Biology

181

116

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Sporadic mutations in the hMeCP2 gene, coding for a protein that preferentially binds symmetrically methylated CpGs, result in the severe neurological disorder Rett syndrome (RTT). In the present work, employing a wide range of experimental approaches, we shed new light on the many levels of MeCP2 interaction with DNA and chromatin. We show that strong methylation-independent as well as methylation-dependent binding by MeCP2 is influenced by DNA length. Although MeCP2 is strictly monomeric in solution, its binding to DNA is cooperative, with dimeric binding strongly correlated with methylation density, and strengthened by nearby A/T repeats. Dimeric binding is abolished in the F155S and R294X severe RTT mutants. MeCP2 also binds chromatin in vitro, resulting in compaction-related changes in nucleosome architecture that resemble the classical zigzag motif induced by histone H1 and considered important for 30-nm-fiber formation. In vivo chromatin binding kinetics and in vitro steady-state nucleosome binding of both MeCP2 and H1 provide strong evidence for competition between MeCP2 and H1 for common binding sites. This suggests that chromatin binding by MeCP2 and H1 in vivo should be viewed in the context of competitive multifactorial regulation.DNA methylation constitutes an important epigenetic component in transcriptional regulation, with methylation generally leading to repression of nearby genes (6). However, the mechanism by which the epigenetic signal is passed to the regulatory machinery is not well understood. Research in this area has been focused on a small family of methyl-CpG binding proteins, best characterized by MeCP2 (19), mutations in which result in Rett syndrome (RTT), a debilitating neurodevelopmental disease in humans (2).A mechanism of MeCP2-mediated gene silencing may involve recruitment of histone deacetylases upon methyl-specific binding (57). However, other mechanisms, which are not necessarily mutually exclusive, such as stabilization of large chromatin loops (29) and promotion of chromatin compaction (51), have also been suggested (14). Studies on in vivo distribution of MeCP2 in nuclei have revealed that, in addition to the expected occupancy of sites of CpG methylation, MeCP2 shows significant binding to unmethylated DNA (71). However, a recent analysis of MeCP2 occupancy has revealed that the genomic distribution of MeCP2 in mammalian neurons closely tracks methyl-CpG density (60). These results highlight our current lack of understanding of key questions pertinent to the binding of MeCP2 to DNA and chromatin. It is especially important, for example, to quantitate the modulation of binding by factors such as methylation density (8,37,48,60) and the presence of adjacent A/T-rich sequences (31) that are reported to influence binding. In the present work, we have used a variety of quantitative approaches to show that, when bound to DNA, MeCP2 exhibits a cooperative monomerdimer equilibrium, which is influenced by DNA length, methylation density, and the presence of nearby A/T repeats.Th...

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“…The angles between DNA helices at junction point were measured as described in our recent paper (28). To measure the angle value lines were drawn over the middle of DNA filaments just near the junction point over the filament sections ca.…”

Section: Methodsmentioning

confidence: 99%

Effect of DNA Supercoiling on the Geometry of Holliday Junctions

2006

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Unusual DNA conformations including cruciforms play an important role in gene regulation and various DNA transactions. Cruciforms are also the models for Holliday junctions, the transient DNA conformations critically involved in DNA homologous and site-specific recombination, repair and replication. Although the conformations of immobile Holliday junctions in linear DNA molecules have been analyzed with use of various techniques, the role of DNA supercoiling has not been studied systematically. We utilized Atomic Force Microscopy (AFM) to visualize cruciform geometry in plasmid DNA with different superhelical densities at various ionic conditions. Both folded and unfolded conformations of the cruciform were identified, and the data showed that DNA supercoiling shifts the equilibrium between folded and unfolded conformations of the cruciform towards the folded one. In topoisomers with low superhelical density the population of folded conformation is 50 to 80 %, depending on ionic strength of the buffer and a type of cation added, whereas in the sample with high superhelical density, this population is as high as 98-100%. The time-lapse studies in aqueous solutions allowed us to observe the conformational transition of the cruciform directly. The time-dependent dynamics of the cruciform correlates with the structural changes revealed by the ensemble-averaged analysis of dry samples. Altogether, the data obtained show directly that DNA supercoiling is the major factor determining the Holliday junction conformation.DNA cruciforms play an important role in regulation of biological processes involving DNA. These structures are formed by inverted repeats and require DNA supercoiling for their stable existence (1). Inverted repeats are associated with origin of replication (1-3). Cruciforms are suggested to be involved in the regulation of gene expression (1,4), and may also play role in nucleosome structure and function (5). In addition, the cruciform is inherent model for Holliday junction, an intermediate in homologous and site-specific recombination (reviewed in (6) (reviewed in (6,14,16)). According to these studies, it can be summarized that the Holiday junction adopts two distinct conformations: folded and unfolded. Unfolded conformation, in which adjacent arms are nearly perpendicular to each other and the structure, has a 4-fold symmetry, and exists at low concentration of metal ions. Folded conformation (or stacked, X-type), in which four arms undergo pairwise coaxial stacking, could be modeled with two duplexes exchanging strands at the junction point. This conformation is stabilized by high ionic strength divalent or polyvalent cations in particular (e.g. Mg2+ cations at the concentration more then 100 μM). Sodium cations also shift equilibrium distribution towards folded conformation though much higher concentrations of cations are required (17). Also, folded conformation of cruciform can be parallel or antiparallel. Synthetic immobile DNA junction exists in antiparallel geometry and no parallel g...

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DNA Strand Arrangement within the SfiI−DNA Complex: Atomic Force Microscopy Analysis

Cited by 41 publications

References 18 publications

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

Molecular Mechanism Underlying RAG1/RAG2 Synaptic Complex Formation

MeCP2 Binds Cooperatively to Its Substrate and Competes with Histone H1 for Chromatin Binding Sites

Effect of DNA Supercoiling on the Geometry of Holliday Junctions

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