RESUMO
The synaptic protein-DNA complexes, formed by specialized proteins that bridge two or more distant sites on DNA, are critically involved in various genetic processes. However, the molecular mechanism by which the protein searches for these sites and how it brings them together is not well understood. Our previous studies directly visualized search pathways used by SfiI, and we identified two pathways, DNA threading and site-bound transfer pathways, specific to the site-search process for synaptic DNA-protein systems. To investigate the molecular mechanism behind these site-search pathways, we assembled complexes of SfiI with various DNA substrates corresponding to different transient states and measured their stability using a single-molecule fluorescence approach. These assemblies corresponded to specific-specific (synaptic), non-specific-non-specific (non-specific), and specific-non-specific (pre-synaptic) SfiI-DNA states. Unexpectedly, an elevated stability in pre-synaptic complexes assembled with specific and non-specific DNA substrates was found. To explain these surprising observations, a theoretical approach that describes the assembly of these complexes and compares the predictions with the experiment was developed. The theory explains this effect by utilizing entropic arguments, according to which, after the partial dissociation, the non-specific DNA template has multiple possibilities of rebinding, effectively increasing the stability. Such difference in the stabilities of SfiI complexes with specific and non-specific DNA explains the utilization of threading and site-bound transfer pathways in the search process of synaptic protein-DNA complexes discovered in the time-lapse AFM experiments.
Assuntos
DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , DNA/química , Proteínas/metabolismo , Ligação Proteica , Replicação do DNARESUMO
The assembly of synaptic protein-DNA complexes by specialized proteins is critical for bringing together two distant sites within a DNA molecule or bridging two DNA molecules. The assembly of such synaptosomes is needed in numerous genetic processes requiring the interactions of two or more sites. The molecular mechanisms by which the protein brings the sites together, enabling the assembly of synaptosomes, remain unknown. Such proteins can utilize sliding, jumping, and segmental transfer pathways proposed for the single-site search process, but none of these pathways explains how the synaptosome assembles. Here we used restriction enzyme SfiI, that requires the assembly of synaptosome for DNA cleavage, as our experimental system and applied time-lapse, high-speed AFM to directly visualize the site search process accomplished by the SfiI enzyme. For the single-site SfiI-DNA complexes, we were able to directly visualize such pathways as sliding, jumping, and segmental site transfer. However, within the synaptic looped complexes, we visualized the threading and site-bound segment transfer as the synaptosome-specific search pathways for SfiI. In addition, we visualized sliding and jumping pathways for the loop dissociated complexes. Based on our data, we propose the site-search model for synaptic protein-DNA systems.
Assuntos
DNA/metabolismo , Proteínas/metabolismo , Sítios de Ligação/fisiologia , Pareamento Cromossômico/fisiologia , Enzimas de Restrição do DNA/metabolismo , Plasmídeos/metabolismo , Ligação Proteica/fisiologia , Sinaptossomos/metabolismoRESUMO
BACKGROUND: Comparative genomics can leverage the vast amount of available genomic sequences to reconstruct and analyze transcriptional regulatory networks in Bacteria, but the efficacy of this approach hinges on the ability to transfer regulatory network information from reference species to the genomes under analysis. Several methods have been proposed to transfer regulatory information between bacterial species, but the paucity and distributed nature of experimental information on bacterial transcriptional networks have prevented their systematic evaluation. RESULTS: We report the compilation of a large catalog of transcription factor-binding sites across Bacteria and its use to systematically benchmark proposed transfer methods across pairs of bacterial species. We evaluate motif- and accuracy-based metrics to assess the results of regulatory network transfer and we identify the precision-recall area-under-the-curve as the best metric for this purpose due to the large class-imbalanced nature of the problem. Methods assuming conservation of the transcription factor-binding motif (motif-based) are shown to substantially outperform those assuming conservation of regulon composition (network-based), even though their efficiency can decrease sharply with increasing phylogenetic distance. Variations of the basic motif-based transfer method do not yield significant improvements in transfer accuracy. Our results indicate that detection of a large enough number of regulated orthologs is critical for network-based transfer methods, but that relaxing orthology requirements does not improve results. Using the transcriptional regulators LexA and Fur as case examples, we also show how DNA-binding domain sequence similarity can yield confounding results as an indicator of transfer efficiency for motif-based methods. CONCLUSIONS: Counter to standard practice, our evaluation of metrics to assess the efficiency of methods for regulatory network information transfer reveals that the area under precision-recall (PR) curves is a more precise and informative metric than that of receiver-operating-characteristic (ROC) curves, confirming similar findings in other class-imbalanced settings. Our systematic assessment of transfer methods reveals that simple approaches to both motif- and network-based transfer of regulatory information provide equal or better results than more elaborate methods. We also show that there are not effective predictors of transfer efficacy, substantiating the long-standing practice of manual curation in comparative genomics analyses.
Assuntos
Bactérias/genética , Redes Reguladoras de Genes/genética , Genômica/métodos , Área Sob a Curva , Sítios de Ligação , Filogenia , Curva ROC , Regulon/genética , Homologia de Sequência de AminoácidosRESUMO
The DNA cytosine deaminase APOBEC3G (A3G) is a two-domain protein that binds single-stranded DNA (ssDNA) largely through its N-terminal domain and catalyzes deamination using its C-terminal domain. A3G is considered an innate immune effector protein, with a natural capacity to block the replication of retroviruses such as HIV and retrotransposons. However, knowledge about its biophysical properties and mechanism of interaction with DNA are still limited. Oligomerization is one of these unclear issues. What is the stoichiometry of the free protein? What are the factors defining the oligomeric state of the protein? How does the protein oligomerization change upon DNA binding? How stable are protein oligomers? We address these questions here using atomic force microscopy (AFM) to directly image A3G protein in a free-state and in complexes with DNA, and using time-lapse AFM imaging to characterize the dynamics of A3G oligomers. We found that the formation of oligomers is an inherent property of A3G and that the yield of oligomers depends on the protein concentration. Oligomerization of A3G in complexes with ssDNA follows a similar pattern: the higher the protein concentrations the larger oligomers sizes. The specificity of A3G binding to ssDNA does not depend on stoichiometry. The binding of large A3G oligomers requires a longer ssDNA substrate; therefore, much smaller oligomers form complexes with short ssDNA. A3G oligomers dissociate spontaneously into monomers and this process primarily occurs through a monomer dissociation pathway.
Assuntos
Citidina Desaminase/química , Desaminase APOBEC-3G , Citidina Desaminase/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Células HEK293 , Humanos , Microscopia de Força Atômica , Tamanho da Partícula , Ligação Proteica , Multimerização Proteica , Imagem com Lapso de TempoRESUMO
UNLABELLED: Locating transcription factor binding sites in genomic sequences is a key step in deciphering transcription networks. Currently available software for site search is mostly server-based, limiting the range and flexibility of this type of analysis. xFITOM is a fully customizable program for locating binding sites in genomic sequences written in C++. Through an easy-to-use interface, xFITOM that allows users an unprecedented degree of flexibility in site search. Among other features,it enables users to define motifs by mixing real sites and IUPAC consensus sequences,to search the annotated sequences of unfinished genomes and to choose among 11 different search algorithms. AVAILABILITY: XFITOM IS AVAILABLE FOR DOWNLOAD AT: http://research.umbc.edu/Ëerill.