Este trabalho foi financiado por uma concessão de CARREIRA NSF (MMH), um Barry M. Goldwater bolsa (FNB) e um Prêmio de Pesquisa ASBMB Graduação (CWD). O clone de sobre-expressão do PBP foi gentilmente cedido pelo Dr. Martin Webb (MRC, UK).

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O exemplo de uma proteína de ligação incompatibilidade DNA aqui descrita ilustra o poder e utilidade dos métodos transientes cinética para estudar os mecanismos de moléculas biológicas. Stopped-flow medições na escala de volume de negócios único momento, desde evidência inequívoca para a ligação rápida e específica de MSH2-MSH6 proteína para um par de bases incompatíveis e formação de uma vida longa complexo DNA-proteína X na reação 9. Além disso, parou de fluxo (e saciar-flow) análi...

<table border="1"><tr><td> Nome de DNA </td><td> Seqüência </td></tr><tr><td> 37 G </td><td> 5&#39;-ATT TCC TTC AGC AGA TAT G TA CCA TGA TAC TTC ACA -3 T &#39; </td></tr><tr><td> 37 T (2-Ap) </td><td> 5&#39;-ATG TGA ATC AGT ATG GTA T Ap T ATC TGC TGA AGG AAA -3 T &#39; </td></tr><tr><td> 37 T </td><td> 5&#39;-ATG TGA ATC AGT ATG GTA T A T ATC TGC TGA AGG -3 AAA T &#39; </td></tr></table>

application of stopped-flow kinetics methods to investigate the mechanism of action of a dna repair protein

Análise cinética transitória é indispensável para a compreensão do funcionamento de macromoléculas biológicas, uma vez que esta abordagem produz informações mecanicista incluindo concentrações sítio ativo e constantes de velocidade intrínseca que governam a função de macromoléculas. Em caso de enzimas, por exemplo, medições estado transiente ou pré-estável identificar e caracterizar os eventos individuais no caminho da reação, enquanto que as medições estado estacionário só o rendimento global de eficiência catalítica e especificidade. Eventos individuais, como a proteína-proteína ou proteína-ligante interações e limitação de taxa de mudanças conformacionais ocorrem frequentemente na escala de tempo de milissegundos, e pode ser medido diretamente pelo stopped-flow e químico-quench métodos de fluxo. Dado um sinal óptico, tais como fluorescência, stopped-flow serve como uma ferramenta poderosa e acessível para monitorar o progresso reação de substrato de ligação para lançamento do produto e volume de negócios 1,2 catalítico. Aqui, relatamos a aplicação de stopped-flow cinética para investigar o mecanismo de ação de MSH2-MSH6, uma proteína de reparo do DNA eucariótico que reconhece pares de bases das disparidades e de inserção / deleção no DNA loops e reparar os sinais de incompatibilidade (MMR) 3-5. Ao fazer isso, MSH2-MSH6 aumenta a precisão da replicação do DNA por três ordens de magnitude (freqüência de erro diminui de ~ 10 -6 a 10 -9 bases), e assim ajuda a preservar a integridade genômica. Não surpreendentemente, com defeito função MSH2-MSH6 humano é associado com câncer de cólon hereditário não-polipose e outros cânceres esporádicos 6-8. A fim de compreender o mecanismo de ação dessa proteína metabólica crítica DNA, estamos investigando a dinâmica de MSH2-MSH6 interação com DNA incompatíveis, bem como a atividade de ATPase que alimenta suas ações no MMR. DNA de ligação é medida pela rápida mistura MSH2-MSH6 com DNA contendo a 2 aminopurina-(2-Ap) adjacente a um fluoróforo G: T incompatibilidade e acompanhamento do aumento resultante em 2 aminopurina-fluorescência em tempo real. DNA dissociação é medido através da mistura pré-formada MSH2-MSH6 G: T (2-Ap) complexo incompatibilidade com o DNA armadilha sem rótulo e diminuir monitoramento de fluorescência ao longo do tempo 9. Pré-ATPase constante cinética de estado são medidos pela mudança de fluorescência de 7-dietilamino-3-((((2-maleimidyl) etil) amino) carbonil) cumarina) marcado Protein Fosfato Binding (MDCC-PBP) em fosfato de ligação ( Pi) liberado pela MSH2-MSH6 seguintes ATP hidrólise 9,10. Os dados revelam ligação rápida de MSH2-MSH6 para um G: Incompatibilidade de T e formação de uma vida longa MSH2-MSH6 G: T complexo, que resulta na supressão da ATP hidrólise e estabilização da proteína em um formulário de ATP-bound . A cinética da reação assegurar um apoio claro para a hipótese de que o ATP-bound MSH2-MSH6 sinais de reparo de DNA em ligação de um par de bases desiguais em dupla hélice. F. Noah Biro e Jie Zhai contribuíram para este trabalho de forma igual.

Watch this Scientific Journal Video about Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein at JoVE.com

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein | Biology | Video

Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

MSH2-MSH6 é responsável por iniciar reparação de erros de replicação de DNA. Aqui nós apresentamos uma abordagem cinética transitória para a compreensão crítica como esta proteína funciona. O relatório ilustra stopped-flow experimentos para medir a ligação DNA acopladas e cinética ATPase subjacente MSH2-MSH6 mecanismo de ação no reparo do DNA.

Aplicação de Métodos Stopped fluxo de Cinética para investigar o mecanismo de ação de uma proteína de reparo do DNA

Transient kinetic analysis is indispensable for understanding the workings of biological macromolecules, since this approach yields mechanistic ...

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18.0K visualizações.  Wesleyan University. MSH2-MSH6 é responsável por iniciar reparação de erros de replicação de DNA. Aqui nós apresentamos uma abordagem cinética transitória para a compreensão crítica como esta proteína funciona. O relatório ilustra stopped-flow experimentos para medir a ligação DNA acopladas e cinética ATPase subjacente MSH2-MSH6 mecanismo de ação no reparo do DNA.

Vídeo: Application of Stopped-flow Kinetics Methods to Investigate the Mechanism of Action of a DNA Repair Protein

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Estudos genéticos de proteínas de reparo do DNA Humano Utilizando Levedura como um sistema modelo

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Biologia

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution of existing proteins, and the assembly of new protein complexes can be stimulated by a variety of DNA lesions and mismatched DNA base pairs. Fluorescence microscopy has been used as a powerful experimental tool for visualizing and quantifying these and other responses to DNA lesions and to monitor DNA replication status within the complex subcellular architecture of a living cell. Translational fusions between fluorescent reporter proteins and components of the DNA replication and repair machinery have been used to determine the cues that target DNA repair proteins to their cognate lesions in vivo and to understand how these proteins are organized within bacterial cells. In addition, transcriptional and translational fusions linked to DNA damage inducible promoters have revealed which cells within a population have activated genotoxic stress responses. In this review, we provide a detailed protocol for using fluorescence microscopy to image the assembly of DNA repair and DNA replication complexes in single bacterial cells. In particular, this work focuses on imaging mismatch repair proteins, homologous recombination, DNA replication and an SOS-inducible protein in Bacillus subtilis. All of the procedures described here are easily amenable for imaging protein complexes in a variety of bacterial species.

Imaging Mismatch Repair and Cellular Responses to DNA Damage in Bacillus subtilis

A detailed protocol is described for imaging the real time formation of DNA repair complexes in Bacillus subtilis cells.

Mismatch Repair imagem e respostas celulares a danos no DNA em Bacillus subtilis</em

Both prokaryotes and eukaryotes respond to DNA damage through a complex set of physiological changes. Alterations in gene expression, the redistribution ...

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), a plant cysteine protease, by real-time label-free analysis using Biacore X100. Several cystatin B variants with point mutations in areas of interaction with papain, are produced. For each cystatin B variant we determine its specific binding concentration using calibration-free concentration analysis (CFCA) and compare the values obtained with total protein concentration as determined by A280. After that, the kinetics of each cystatin B variant binding to papain is measured using single-cycle kinetics (SCK). We show that one of the four cystatin B variants we examine is only partially active for binding. This partial activity, revealed by CFCA, translates to a significant difference in the association rate constant (ka) and affinity (KD), compared to the values calculated using total protein concentration. Using CFCA in combination with kinetic analysis in a structure-function study contributes to obtaining reliable results, and helps to make the right interpretation of the interaction mechanism.

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

We apply label-free protein interaction analysis using Biacore X100 for structure-function analysis of the binding of several cystatin B mutants to papain through kinetic characterization. Calibration-free concentration analysis (CFCA) measures the concentration of protein with retained binding activity without the need for a standard curve. We show that confirmation of concentrations using CFCA increases the reliability of the kinetic analysis and that kinetic constants can reliably be determined even if the activity of a recombinant protein is reduced.

A importância da concentração de proteína correta para Cinética e Determinação Affinity na estrutura-função Análise

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

Oxidative stress is an unavoidable byproduct of aerobic life. Molecular oxygen is essential for terrestrial metabolism, but it also takes part in many damaging reactions within living organisms. The combination of aerobic metabolism and iron, which is another vital compound for life, is enough to produce radicals through Fenton chemistry and degrade cellular components. DNA degradation is arguably the most damaging process involving intracellular radicals, as DNA repair is far from trivial. The assay presented in this article offers a quantitative technique to measure and visualize the effect of molecules and enzymes on radical-mediated DNA damage.
The DNA protection assay is a simple, quick, and robust tool for the in vitro characterization of the protective properties of proteins or chemicals. It involves exposing DNA to a damaging oxidative reaction and adding varying concentrations of the compound of interest. The reduction or increase of DNA damage as a function of compound concentration is then visualized using gel electrophoresis. In this article we demonstrate the technique of the DNA protection assay by measuring the protective properties of the DNA-binding protein from starved cells (Dps). Dps is a mini-ferritin that is utilized by more than 300 bacterial species to powerfully combat environmental stressors. Here we present the Dps purification protocol and the optimized assay conditions for evaluating DNA protection by Dps.

Application of an In vitro DNA Protection Assay to Visualize Stress Mediation Properties of the Dps Protein

The DNA-binding protein from starved cells (Dps) plays a crucial role in combating bacterial stress. This article discusses the purification of E. coli Dps and the protocol for an in vitro assay demonstrating Dps-mediated protection of DNA from degradation by reactive oxygen species.

A aplicação de um<em&gt; In vitro</em&gt; DNA Assay Proteção ao Visualize Propriedades Mediação stress do Dps Protein

Oxidative stress is an unavoidable byproduct of  aerobic life.  Molecular oxygen is  essential for terrestrial metabolism, but it also takes part in many ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

Imagens em tempo real de uma única Engineered RNA transcritos em células vivas usando raciométrica Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Development of Inhibitors of Protein-protein Interactions through REPLACE: Application to the Design and Development Non-ATP Competitive CDK Inhibitors

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by providing improved methodology for the identification of inhibitors for such binding sites and which represent the majority of potential drug targets. The main goal of this paper is to provide a methodological overview of the use and application of the REPLACE strategy which involves computational and synthetic chemistry approaches. REPLACE is exemplified through its application to the development of non-ATP competitive cyclin dependent kinases (CDK) inhibitors as anti-tumor therapeutics. CDKs are frequently deregulated in cancer and hence are considered as important targets for drug development. Inhibition of CDK2/cyclin A in S phase has been reported to promote selective apoptosis of cancer cells in a p53 independent manner through the E2F1 pathway. Targeting the protein-protein interaction at the cyclin binding groove (CBG) is an approach which will allow the specific inhibition of cell cycle over transcriptional CDKs. The CBG is recognized by a consensus sequence derived from CDK substrates and tumor suppressor proteins termed the cyclin binding motif (CBM). The CBM has previously been optimized to an octapeptide from p21Waf (HAKRRIF) and then further truncated to a pentapeptide retaining sufficient activity (RRLIF). Peptides in general are not cell permeable, are metabolically unstable and therefore the REPLACE (REplacement with Partial Ligand Alternatives through Computational Enrichment) strategy has been applied in order to generate more drug-like inhibitors. The strategy begins with the design of Fragment ligated inhibitory peptides (FLIPs) that selectively inhibit cell cycle CDK/cyclin complexes. FLIPs were generated by iteratively replacing residues of HAKRRLIF/RRLIF with fragment like small molecules (capping groups), starting from the N-terminus (Ncaps), followed by replacement on the C-terminus. These compounds are starting points for the generation of non-ATP competitive CDK inhibitors as anti-tumor therapeutics.

We describe implementation of the REPLACE strategy for targeting protein-protein interactions. REPLACE is an iterative strategy involving synthetic and computational approaches for the conversion of optimized peptidic inhibitors into drug like molecules.

Desenvolvimento de inibidores de interacções proteína-proteína através REPLACE: Aplicação para a concepção e desenvolvimento não-ATP inibidores de CDK Competitiva

REPLACE is a unique strategy developed to more effectively target protein-protein interactions (PPIs). It aims to expand available drug target space by ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Ensaio para imunoprecipitação da cromatina a identificação de Arabidopsis interações proteína-ADN<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Amplification, Next-generation Sequencing, and Genomic DNA Mapping of Retroviral Integration Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of diverse libraries of retroviral integration sites using ligation-mediated PCR (LM-PCR) amplification and next-generation sequencing (NGS), (2) mapping the genomic location of each virus-host junction using BEDTools, and (3) analyzing the data for statistical relevance. Genomic DNA extracted from infected cells is fragmented by digestion with restriction enzymes or by sonication. After suitable DNA end-repair, double-stranded linkers are ligated onto the DNA ends, and semi-nested PCR is conducted using primers complementary to both the long terminal repeat (LTR) end of the virus and the ligated linker DNA. The PCR primers carry sequences required for DNA clustering during NGS, negating the requirement for separate adapter ligation. Quality control (QC) is conducted to assess DNA fragment size distribution and adapter DNA incorporation prior to NGS. Sequence output files are filtered for LTR-containing reads, and the sequences defining the LTR and the linker are cropped away. Trimmed host cell sequences are mapped to a reference genome using BLAT and are filtered for minimally 97% identity to a unique point in the reference genome. Unique integration sites are scrutinized for adjacent nucleotide (nt) sequence and distribution relative to various genomic features. Using this protocol, integration site libraries of high complexity can be constructed from genomic DNA in three days. The entire protocol that encompasses exogenous viral infection of susceptible tissue culture cells to integration site analysis can therefore be conducted in approximately one to two weeks. Recent applications of this technology pertain to longitudinal analysis of integration sites from HIV-infected patients.

We describe a protocol for amplifying retroviral integration sites from the genomic DNA of infected cells, sequencing the amplified virus-host junctions, and then mapping these sequences to a reference genome. We also describe techniques to quantify the distribution of integration sites relative to various genomic annotations using BEDTools.

Amplificação, sequenciamento de próxima geração, e Genomic DNA Mapeamento das retrovirais Integração Sites

Retroviruses exhibit signature integration preferences on both the local and global scales. Here, we present a detailed protocol for (1) generation of ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

A visualização de tethered-grande superfície moléculas de ADN com um ADN de proteína fluorescente Binding Peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Avançadas técnicas de microscopia Confocal para estudar interações da proteína-proteína e cinética de lesões do ADN

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...