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1.
Nat Commun ; 14(1): 7072, 2023 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-37923737

RESUMO

Retrovirus integration into a host genome is essential for productive infections. The integration strand transfer reaction is catalyzed by a nucleoprotein complex (Intasome) containing the viral integrase (IN) and the reverse transcribed (RT) copy DNA (cDNA). Previous studies suggested that DNA target-site recognition limits intasome integration. Using single molecule Förster resonance energy transfer (smFRET), we show prototype foamy virus (PFV) intasomes specifically bind to DNA strand breaks and gaps. These break and gap DNA discontinuities mimic oxidative base excision repair (BER) lesion-processing intermediates that have been shown to affect retrovirus integration in vivo. The increased DNA binding events targeted strand transfer to the break/gap site without inducing substantial intasome conformational changes. The major oxidative BER substrate 8-oxo-guanine as well as a G/T mismatch or +T nucleotide insertion that typically introduce a bend or localized flexibility into the DNA, did not increase intasome binding or targeted integration. These results identify DNA breaks or gaps as modulators of dynamic intasome-target DNA interactions that encourage site-directed integration.


Assuntos
DNA Viral , Spumavirus , DNA Viral/metabolismo , Integrases/metabolismo , Retroviridae/genética , Retroviridae/metabolismo , Spumavirus/genética , Spumavirus/metabolismo , DNA Complementar , Integração Viral
2.
Nat Commun ; 13(1): 5808, 2022 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-36192430

RESUMO

Highly conserved MutS and MutL homologs operate as protein dimers in mismatch repair (MMR). MutS recognizes mismatched nucleotides forming ATP-bound sliding clamps, which subsequently load MutL sliding clamps that coordinate MMR excision. Several MMR models envision static MutS-MutL complexes bound to mismatched DNA via a positively charged cleft (PCC) located on the MutL N-terminal domains (NTD). We show MutL-DNA binding is undetectable in physiological conditions. Instead, MutS sliding clamps exploit the PCC to position a MutL NTD on the DNA backbone, likely enabling diffusion-mediated wrapping of the remaining MutL domains around the DNA. The resulting MutL sliding clamp enhances MutH endonuclease and UvrD helicase activities on the DNA, which also engage the PCC during strand-specific incision/excision. These MutS clamp-loader progressions are significantly different from the replication clamp-loaders that attach the polymerase processivity factors ß-clamp/PCNA to DNA, highlighting the breadth of mechanisms for stably linking crucial genome maintenance proteins onto DNA.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Reparo do DNA , Endonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas MutL/genética , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Nucleotídeos , Antígeno Nuclear de Célula em Proliferação/metabolismo
3.
J Biol Chem ; 298(11): 102505, 2022 11.
Artigo em Inglês | MEDLINE | ID: mdl-36126773

RESUMO

MutS homologs (MSHs) are highly conserved core components of DNA mismatch repair. Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Here, we have expanded on previous bulk biochemical studies to examine the stability, lifetime, and kinetics of bacterial and human MSH sliding clamps on mismatched DNA using surface plasmon resonance and single-molecule analysis of fluorescently labeled proteins. We found that ATP-bound MSH complexes bound to blocked-end or very long mismatched DNAs were extremely stable over a range of ionic conditions. These observations underpinned the development of a high-throughput Förster resonance energy transfer system that specifically detects the formation of MSH sliding clamps on mismatched DNA. The Förster resonance energy transfer system is capable of distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 and appears suitable for chemical inhibitor screens. Taken together, our results provide additional insight into MSH sliding clamps as well as methods to distinguish their functions in mismatch repair.


Assuntos
Proteínas de Escherichia coli , Proteína MutS de Ligação de DNA com Erro de Pareamento , Humanos , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , DNA/metabolismo , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Proteína 2 Homóloga a MutS/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteínas MutS/genética , Ligação Proteica
4.
Sci Rep ; 12(1): 4082, 2022 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-35260723

RESUMO

The Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2), also known as 2019 novel coronavirus (2019-nCoV), is a highly infectious RNA virus. A percentage of patients develop coronavirus disease 2019 (COVID-19) after infection, whose symptoms include fever, cough, shortness of breath and fatigue. Acute and life-threatening respiratory symptoms are experienced by 10-20% of symptomatic patients, particularly those with underlying medical conditions. One of the main challenges in the containment of COVID-19 is the identification and isolation of asymptomatic/pre-symptomatic individuals. A number of molecular assays are currently used to detect SARS-CoV-2. Many of them can accurately test hundreds or even thousands of patients every day. However, there are presently no testing platforms that enable more than 10,000 tests per day. Here, we describe the foundation for the REcombinase Mediated BaRcoding and AmplificatioN Diagnostic Tool (REMBRANDT), a high-throughput Next Generation Sequencing-based approach for the simultaneous screening of over 100,000 samples per day. The REMBRANDT protocol includes direct two-barcoded amplification of SARS-CoV-2 and control amplicons using an isothermal reaction, and the downstream library preparation for Illumina sequencing and bioinformatics analysis. This protocol represents a potentially powerful approach for community screening of COVID-19 that may be modified for application to any infectious or non-infectious genome.


Assuntos
COVID-19/diagnóstico , Proteínas de Ligação a DNA/metabolismo , Proteínas de Membrana/metabolismo , Técnicas de Amplificação de Ácido Nucleico/métodos , SARS-CoV-2/genética , Proteínas Virais/metabolismo , COVID-19/virologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Programas de Rastreamento , RNA Viral/análise , RNA Viral/metabolismo , SARS-CoV-2/isolamento & purificação
5.
Phys Rev E ; 103(5-1): 052404, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-34134264

RESUMO

Protein complexes involved in DNA mismatch repair diffuse along dsDNA as sliding clamps in order to locate a hemimethylated incision site. They have been observed to use a dissociative mechanism, in which two proteins, while continuously remaining attached to the DNA, sometimes associate into a single complex sliding on the DNA and sometimes dissociate into two independently sliding proteins. Here, we study the probability that these complexes locate a given target site via a semi-analytic, Monte Carlo calculation that tracks the association and dissociation of the sliding complexes. We compare such probabilities to those obtained using a nondissociative diffusive scan in the space of physically realistic diffusion constants, hemimethylated site distances, and total search times to determine the regions in which dissociative searching is more or less efficient than nondissociative searching. We conclude that the dissociative search mechanism is advantageous in the majority of the physically realistic parameter space, suggesting that the dissociative search mechanism confers an evolutionary advantage.


Assuntos
Reparo de Erro de Pareamento de DNA , Difusão
6.
Front Mol Biosci ; 8: 662331, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34055882

RESUMO

Retroviruses are obligate intracellular parasites that must integrate a copy of the viral genome into the host DNA. The integration reaction is performed by the viral enzyme integrase in complex with the two ends of the viral cDNA genome and yields an integrated provirus. Retroviral vector particles are attractive gene therapy delivery tools due to their stable integration. However, some retroviral integration events may dysregulate host oncogenes leading to cancer in gene therapy patients. Multiple strategies to target retroviral integration, particularly to genetic safe harbors, have been tested with limited success. Attempts to target integration may be limited by the multimerization of integrase or the presence of host co-factors for integration. Several retroviral integration complexes have evolved a mechanism of tethering to chromatin via a host protein. Integration host co-factors bind chromatin, anchoring the complex and allowing integration. The tethering factor allows for both close proximity to the target DNA and specificity of targeting. Each retrovirus appears to have distinct preferences for DNA sequence and chromatin features at the integration site. Tethering factors determine the preference for chromatin features, but do not affect the subtle sequence preference at the integration site. The sequence preference is likely intrinsic to the integrase protein. New developments may uncouple the requirement for a tethering factor and increase the ability to redirect retroviral integration.

7.
Curr Biol ; 31(6): R293-R296, 2021 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-33756142

RESUMO

State-of-the-art genetic and cellular studies uniquely implicate the S. cerevisiae Pms1 endonuclease (human PMS2) and ExoI as the major components that produce and/or maintain the strand-specific nicks that precisely direct mismatch repair.


Assuntos
Reparo de Erro de Pareamento de DNA , Proteínas de Saccharomyces cerevisiae , Humanos , Proteínas MutL , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
J Biol Chem ; 296: 100550, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33744295

RESUMO

Retroviral integrases must navigate host DNA packaged as chromatin during integration of the viral genome. Prototype foamy virus (PFV) integrase (IN) forms a tetramer bound to two viral DNA (vDNA) ends in a complex termed an intasome. PFV IN consists of four domains: the amino terminal extension domain (NED), amino terminal domain (NTD), catalytic core domain (CCD), and carboxyl terminal domain (CTD). The domains of the two inner IN protomers have been visualized, as well as the CCDs of the two outer IN protomers. However, the roles of the amino and carboxyl terminal domains of the PFV intasome outer subunits during integration to a nucleosome target substrate are not clear. We used the well-characterized 601 nucleosome to assay integration activity as well as intasome binding. PFV intasome integration to 601 nucleosomes occurs in clusters at four independent sites. We find that the outer protomer NED and NTD domains have no significant effects on integration efficiency, site selection, or binding. The CTDs of the outer PFV intasome subunits dramatically affect nucleosome binding but have little effect on total integration efficiency. The outer PFV IN CTDs did significantly alter the integration efficiency at one site. Histone tails also significantly affect intasome binding, but have little impact on PFV integration efficiency or site selection. These results indicate that binding to nucleosomes does not correlate with integration efficiency and suggests most intasome-binding events are unproductive.


Assuntos
Histonas/metabolismo , Integrases/metabolismo , Nucleossomos/metabolismo , Spumavirus/metabolismo , Proteínas Virais/metabolismo , Integração Viral , Domínio Catalítico , Cromatina/genética , Cromatina/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Genoma Viral , Humanos , Integrases/genética , Multimerização Proteica , Spumavirus/genética , Spumavirus/crescimento & desenvolvimento , Proteínas Virais/química , Proteínas Virais/genética
9.
Proc Natl Acad Sci U S A ; 118(9)2021 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-33619096

RESUMO

The pathogenic consequences of 369 unique human HsMLH1 missense variants has been hampered by the lack of a detailed function in mismatch repair (MMR). Here single-molecule images show that HsMSH2-HsMSH6 provides a platform for HsMLH1-HsPMS2 to form a stable sliding clamp on mismatched DNA. The mechanics of sliding clamp progression solves a significant operational puzzle in MMR and provides explicit predictions for the distribution of clinically relevant HsMLH1 missense mutations.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Proteínas de Ligação a DNA/genética , DNA/genética , Proteína 1 Homóloga a MutL/genética , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Sítios de Ligação , Neoplasias Colorretais Hereditárias sem Polipose/metabolismo , Neoplasias Colorretais Hereditárias sem Polipose/patologia , DNA/química , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Humanos , Modelos Moleculares , Proteína 1 Homóloga a MutL/química , Proteína 1 Homóloga a MutL/metabolismo , Proteína 2 Homóloga a MutS/química , Proteína 2 Homóloga a MutS/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas
10.
Nat Commun ; 10(1): 5294, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757945

RESUMO

A shared paradigm of mismatch repair (MMR) across biology depicts extensive exonuclease-driven strand-specific excision that begins at a distant single-stranded DNA (ssDNA) break and proceeds back past the mismatched nucleotides. Historical reconstitution studies concluded that Escherichia coli (Ec) MMR employed EcMutS, EcMutL, EcMutH, EcUvrD, EcSSB and one of four ssDNA exonucleases to accomplish excision. Recent single-molecule images demonstrated that EcMutS and EcMutL formed cascading sliding clamps on a mismatched DNA that together assisted EcMutH in introducing ssDNA breaks at distant newly replicated GATC sites. Here we visualize the complete strand-specific excision process and find that long-lived EcMutL sliding clamps capture EcUvrD helicase near the ssDNA break, significantly increasing its unwinding processivity. EcSSB modulates the EcMutL-EcUvrD unwinding dynamics, which is rarely accompanied by extensive ssDNA exonuclease digestion. Together these observations are consistent with an exonuclease-independent MMR strand excision mechanism that relies on EcMutL-EcUvrD helicase-driven displacement of ssDNA segments between adjacent EcMutH-GATC incisions.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Helicases/fisiologia , Reparo de Erro de Pareamento de DNA/fisiologia , Proteínas de Escherichia coli/fisiologia , Escherichia coli/fisiologia , Proteínas MutL/fisiologia , DNA Helicases/metabolismo , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microscopia de Fluorescência , Proteínas MutL/metabolismo , Imagem Individual de Molécula
11.
PLoS One ; 14(3): e0212764, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30865665

RESUMO

Eukaryotic DNA binding proteins must access genomic DNA that is packaged into chromatin in vivo. During a productive infection, retroviral integrases (IN) must similarly interact with chromatin to integrate the viral cDNA genome. Here we examine the role of nucleosome DNA unwrapping in the retroviral integrase search for a target site. These studies utilized PFV intasomes that are comprised of a tetramer of PFV IN with two oligomers mimicking the viral cDNA ends. Modified recombinant human histones were used to generate nucleosomes with increased unwrapping rates at different DNA regions. These modifications included the acetylmimetic H3(K56Q) and the chemically engineered H4(K77ac, K79ac). While transcription factors and DNA damage sensors may search nucleosome bound DNA during transient unwrapping, PFV intasome mediated integration appears to be unaffected by increased nucleosome unwrapping. These studies suggest PFV intasomes do not utilize nucleosome unwrapping to search nucleosome targets.


Assuntos
DNA Viral/metabolismo , Genoma Viral , Nucleossomos/metabolismo , Spumavirus/metabolismo , Integração Viral/fisiologia , Sistema Livre de Células/química , Sistema Livre de Células/metabolismo , DNA Viral/química , Histonas/química , Histonas/metabolismo , Humanos , Nucleossomos/química , Spumavirus/química
12.
J Biol Chem ; 293(37): 14285-14294, 2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30072380

RESUMO

Sliding clamps on DNA consist of evolutionarily conserved enzymes that coordinate DNA replication, repair, and the cellular DNA damage response. MutS homolog (MSH) proteins initiate mismatch repair (MMR) by recognizing mispaired nucleotides and in the presence of ATP form stable sliding clamps that randomly diffuse along the DNA. The MSH sliding clamps subsequently load MutL homolog (MLH/PMS) proteins that form a second extremely stable sliding clamp, which together coordinate downstream MMR components with the excision-initiation site that may be hundreds to thousands of nucleotides distant from the mismatch. Specific or nonspecific binding of other proteins to the DNA between the mismatch and the distant excision-initiation site could conceivably obstruct the free diffusion of these MMR sliding clamps, inhibiting their ability to initiate repair. Here, we employed bulk biochemical analysis, single-molecule fluorescence imaging, and mathematical modeling to determine how sliding clamps might overcome such hindrances along the DNA. Using both bacterial and human MSH proteins, we found that increasing the number of MSH sliding clamps on a DNA decreased the association of the Escherichia coli transcriptional repressor LacI to its cognate promoter LacO. Our results suggest a simple mechanism whereby thermal diffusion of MSH sliding clamps along the DNA alters the association kinetics of other DNA-binding proteins over extended distances. These observations appear generally applicable to any stable sliding clamp that forms on DNA.


Assuntos
DNA Bacteriano/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Thermus/metabolismo , Trifosfato de Adenosina/metabolismo , Pareamento Incorreto de Bases , Modelos Teóricos , Ligação Proteica , Ressonância de Plasmônio de Superfície
14.
Anal Biochem ; 556: 78-84, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29932890

RESUMO

Single-molecule (SM) microscopy is a powerful tool capable of visualizing individual molecules and events in real time. SM imaging may rely on proteins or nucleic acids labelled with a fluorophore. Unfortunately photobleaching of fluorophores leads to irreversible loss of signal, impacting the collection of data from SM experiments. Trace amounts of dissolved oxygen (O2) are the main cause of photobleaching. Oxygen scavenging systems (OSS) have been developed that decrease dissolved O2. Commercial OSS enzyme preparations are frequently contaminated with nucleases that damage nucleic acid substrates. In this protocol, we purify highly active Pseudomonas putida protocatechuate 3,4-dioxygenase (PCD) without nuclease contaminations. Quantitation of Cy3 photostability revealed that PCD with its substrate protocatechuic acid (PCA) increased the fluorophore half-life 100-fold. This low cost purification method of recombinant PCD yields an enzyme superior to commercially available OSS that is effectively free of nuclease activity.


Assuntos
Proteínas de Bactérias , Expressão Gênica , Hidroxibenzoatos/química , Imagem Óptica , Protocatecoate-3,4-Dioxigenase , Pseudomonas putida , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Desoxirribonucleases , Estabilidade Enzimática , Oxigênio/química , Protocatecoate-3,4-Dioxigenase/biossíntese , Protocatecoate-3,4-Dioxigenase/química , Protocatecoate-3,4-Dioxigenase/genética , Protocatecoate-3,4-Dioxigenase/isolamento & purificação , Pseudomonas putida/enzimologia , Pseudomonas putida/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação
15.
J Mol Biol ; 430(22): 4456-4468, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29864444

RESUMO

DNA mismatch repair (MMR) is a DNA excision-resynthesis process that principally enhances replication fidelity. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologs initiate MMR and in higher eukaryotes act as DNA damage sensors that can trigger apoptosis. MSH proteins recognize mismatched nucleotides, whereas the MLH/PMS proteins mediate multiple interactions associated with downstream MMR events including strand discrimination and strand-specific excision that are initiated at a significant distance from the mismatch. Remarkably, the biophysical functions of the MLH/PMS proteins have been elusive for decades. Here we consider recent observations that have helped to define the mechanics of MLH/PMS proteins and their role in choreographing MMR. We highlight the stochastic nature of DNA interactions that have been visualized by single-molecule analysis and the plasticity of protein complexes that employ thermal diffusion to complete the progressions of MMR.


Assuntos
DNA/metabolismo , Proteínas MutL/metabolismo , Imagem Individual de Molécula/métodos , Animais , Reparo de Erro de Pareamento de DNA , Humanos , Cinética , Transdução de Sinais , Processos Estocásticos
16.
J Mol Biol ; 430(22): 4469-4480, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29792877

RESUMO

DNA mismatch repair (MMR) corrects DNA base-pairing errors that occur during DNA replication. MMR catalyzes strand-specific DNA degradation and resynthesis by dynamic molecular coordination of sequential downstream pathways. The temporal and mechanistic order of molecular events is essential to insure interactions in MMR that occur over long distances on the DNA. Biophysical real-time studies of highly conserved components on mismatched DNA have shed light on the mechanics of MMR. Single-molecule imaging has visualized stochastically coordinated MMR interactions that are based on thermal fluctuation-driven motions. In this review, we describe the role of diffusivity and stochasticity in MMR beginning with mismatch recognition through strand-specific excision. We conclude with a perspective of the possible research directions that should solve the remaining questions in MMR.


Assuntos
DNA/metabolismo , Complexos Multiproteicos/metabolismo , Animais , Fenômenos Biofísicos , Reparo de Erro de Pareamento de DNA , Difusão , Humanos , Processos Estocásticos , Termodinâmica
17.
J Biol Chem ; 293(24): 9473-9485, 2018 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-29674319

RESUMO

Conformations adopted by long stretches of single-stranded DNA (ssDNA) are of central interest in understanding the architecture of replication forks, R loops, and other structures generated during DNA metabolism in vivo This is particularly so if the ssDNA consists of short nucleotide repeats. Such studies have been hampered by the lack of defined substrates greater than ∼150 nt and the absence of high-resolution biophysical approaches. Here we describe the generation of very long ssDNA consisting of the mammalian telomeric repeat (5'-TTAGGG-3') n , as well as the interrogation of its structure by EM and single-molecule magnetic tweezers (smMT). This repeat is of particular interest because it contains a run of three contiguous guanine residues capable of forming G quartets as ssDNA. Fluorescent-dye exclusion assays confirmed that this G-strand ssDNA forms ubiquitous G-quadruplex folds. EM revealed thick bead-like filaments that condensed the DNA ∼12-fold. The bead-like structures were 5 and 8 nm in diameter and linked by thin filaments. The G-strand ssDNA displayed initial stability to smMT force extension that ultimately released in steps that were multiples ∼28 nm at forces between 6 and 12 pN, well below the >20 pN required to unravel G-quadruplexes. Most smMT steps were consistent with the disruption of the beads seen by EM. Binding by RAD51 distinctively altered the force extension properties of the G-strand ssDNA, suggesting a stochastic G-quadruplex-dependent condensation model that is discussed.


Assuntos
DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Quadruplex G , Sequência de Bases , DNA de Cadeia Simples/metabolismo , Humanos , Ligação Proteica , Rad51 Recombinase/metabolismo , Telômero/química , Telômero/metabolismo , Telômero/ultraestrutura
18.
Oncotarget ; 9(16): 12554-12561, 2018 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-29560090

RESUMO

Non-steroidal anti-inflammatory drugs (NSAIDs) exhibit anti-neoplastic (chemoprevention) activity for sporadic cancers and the hereditary cancer predisposition Lynch syndrome (LS/HNPCC). However, the mechanism of NSAID tumor suppression has remained enigmatic. Defects in the core mismatch repair (MMR) genes MSH2 and MLH1 are the principal drivers of LS/HNPCC. Previous work has demonstrated that the villin-Cre+/-Msh2flox/flox (VpC-Msh2) mouse is a reliable model for LS/HNPCC intestinal tumorigenesis, which is significantly suppressed by treatment with the NSAID aspirin (ASA) similar to human chemoprevention. Here we show that including a TGFß receptor type-II (Tgfß-RII) mutation in the VpC-Msh2 mouse (villin-Cre+/-Msh2flox/floxTgfß-RIIflox/flox ) completely eliminates NSAID tumor suppression. These results provide strong genetic evidence that TGFß signaling and/or effectors participate in NSAID-dependent anti-neoplastic processes and provide fresh avenues for understanding NSAID chemoprevention and resistance.

19.
Nucleic Acids Res ; 45(2): 685-698, 2017 01 25.
Artigo em Inglês | MEDLINE | ID: mdl-27738136

RESUMO

Wrapping of genomic DNA into nucleosomes poses thermodynamic and kinetic barriers to biological processes such as replication, transcription, repair and recombination. Previous biochemical studies have demonstrated that in the presence of adenosine triphosphate (ATP) the human RAD51 (HsRAD51) recombinase can form a nucleoprotein filament (NPF) on double-stranded DNA (dsDNA) that is capable of unwrapping the nucleosomal DNA from the histone octamer (HO). Here, we have used single molecule Förster Resonance Energy Transfer (smFRET) to examine the real time nucleosome dynamics in the presence of the HsRAD51 NPF. We show that oligomerization of HsRAD51 leads to stepwise, but stochastic unwrapping of the DNA from the HO in the presence of ATP. The highly reversible dynamics observed in single-molecule trajectories suggests an antagonistic mechanism between HsRAD51 binding and rewrapping of the DNA around the HO. These stochastic dynamics were independent of the nucleosomal DNA sequence or the asymmetry created by the presence of a linker DNA. We also observed sliding and rotational oscillations of the HO with respect to the nucleosomal DNA. These studies underline the dynamic nature of even tightly associated protein-DNA complexes such as nucleosomes.


Assuntos
Histonas/metabolismo , Nucleossomos/metabolismo , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/genética , DNA/metabolismo , Replicação do DNA , Histonas/química , Humanos , Hidrólise , Modelos Biológicos , Nucleoproteínas/metabolismo , Ligação Proteica , Multimerização Proteica
20.
Nature ; 539(7630): 583-587, 2016 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-27851738

RESUMO

Mismatched nucleotides arise from polymerase misincorporation errors, recombination between heteroallelic parents and chemical or physical DNA damage. Highly conserved MutS (MSH) and MutL (MLH/PMS) homologues initiate mismatch repair and, in higher eukaryotes, act as DNA damage sensors that can trigger apoptosis. Defects in human mismatch repair genes cause Lynch syndrome or hereditary non-polyposis colorectal cancer and 10-40% of related sporadic tumours. However, the collaborative mechanics of MSH and MLH/PMS proteins have not been resolved in any organism. We visualized Escherichia coli (Ec) ensemble mismatch repair and confirmed that EcMutS mismatch recognition results in the formation of stable ATP-bound sliding clamps that randomly diffuse along the DNA with intermittent backbone contact. The EcMutS sliding clamps act as a platform to recruit EcMutL onto the mismatched DNA, forming an EcMutS-EcMutL search complex that then closely follows the DNA backbone. ATP binding by EcMutL establishes a second long-lived DNA clamp that oscillates between the principal EcMutS-EcMutL search complex and unrestricted EcMutS and EcMutL sliding clamps. The EcMutH endonuclease that targets mismatch repair excision only binds clamped EcMutL, increasing its DNA association kinetics by more than 1,000-fold. The assembly of an EcMutS-EcMutL-EcMutH search complex illustrates how sequential stable sliding clamps can modulate one-dimensional diffusion mechanics along the DNA to direct mismatch repair.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA/metabolismo , Difusão , Proteínas de Escherichia coli/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas MutL/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Trifosfato de Adenosina/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Cinética , Complexos Multiproteicos/química , Proteínas MutL/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Transporte Proteico , Imagem Individual de Molécula
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