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1.
Chem Res Toxicol ; 31(8): 697-711, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-30004685

RESUMO

Specialized DNA damage-bypass Y-family DNA polymerases contribute to cancer prevention by providing cellular tolerance to DNA damage that can lead to mutations and contribute to cancer progression by increasing genomic instability. Y-family polymerases can also bypass DNA adducts caused by chemotherapy agents. One of the four human Y-family DNA polymerases, DNA polymerase (pol) κ, has been shown to be specific for bypass of minor groove adducts and inhibited by major groove adducts. In addition, mutations in the gene encoding pol κ are associated with different types of cancers as well as with chemotherapy responses. We characterized nine variants of pol κ whose identity was inferred from cancer-associated single nucleotide polymorphisms for polymerization activity on undamaged and damaged DNA, their abilities to extend from mismatched or damaged base pairs at primer termini, and overall stability and dynamics. We find that these pol κ variants generally fall into three categories: similar activity to wild-type (WT) pol κ (L21F, I39T, P169T, F192C, and E292K), more active than WT pol κ (S423R), and less active than pol κ (R219I, R298H, and Y432S). Of these, only pol κ variants R298H and Y432S had markedly reduced thermal stability. Molecular dynamics (MD) simulations with undamaged DNA revealed that the active variant F192C and more active variant S423R with either correct or incorrect incoming nucleotide mimic WT pol κ with the correct incoming nucleotide, whereas the less active variants R219I, R298H, and Y432S with the correct incoming nucleotide mimic WT pol κ with the incorrect incoming nucleotide. Thus, the observations from MD simulations suggest a possible explanation for the observed experimental results that pol κ adopts specific active and inactive conformations that depend on both the protein variant and the identity of the DNA adduct.


Assuntos
DNA Polimerase Dirigida por DNA/genética , Neoplasias/enzimologia , Pareamento de Bases , Humanos , Simulação de Dinâmica Molecular , Polimorfismo de Nucleotídeo Único , Moldes Genéticos
2.
DNA Repair (Amst) ; 12(9): 733-40, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23830898

RESUMO

The advent of complete-genome genotyping across phenotype cohorts has provided a rich source of information for bioinformaticians. However the search for SNPs from this data is generally performed on a study-by-study case without any specific hypothesis of the location for SNPs that are predictive for the phenotype. We have designed a method whereby very large SNP lists (several gigabytes in size), combining several genotyping studies at once, can be sorted and traced back to their ultimate consequence in protein structure. Given a working hypothesis, researchers are able to easily search whole genome genotyping data for SNPs that link genetic locations to phenotypes. This allows a targeted search for correlations between phenotypes and potentially relevant systems, rather than utilizing statistical methods only. HyDn-SNP-S returns results that are less data dense, allowing more thorough analysis, including haplotype analysis. We have applied our method to correlate DNA polymerases to cancer phenotypes using four of the available cancer databases in dbGaP. Logistic regression and derived haplotype analysis indicates that ~80SNPs, previously overlooked, are statistically significant. Derived haplotypes from this work link POLL to breast cancer and POLG to prostate cancer with an increase in incidence of 3.01- and 9.6-fold, respectively. Molecular dynamics simulations on wild-type and one of the SNP mutants from the haplotype of POLL provide insights at the atomic level on the functional impact of this cancer related SNP. Furthermore, HyDn-SNP-S has been designed to allow application to any system. The program is available upon request from the authors.


Assuntos
Polimorfismo de Nucleotídeo Único , Software , Algoritmos , Substituição de Aminoácidos , Domínio Catalítico , DNA Polimerase beta/química , DNA Polimerase beta/genética , Mineração de Dados/métodos , Bases de Dados de Ácidos Nucleicos , Genoma Humano , Estudo de Associação Genômica Ampla , Humanos , Modelos Genéticos , Simulação de Dinâmica Molecular , Neoplasias/genética , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína
3.
Biophys J ; 105(2): 494-501, 2013 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-23870270

RESUMO

Clostridium difficile (C. diff) is one of the most common and most severe hospital-acquired infections; its consequences range from lengthened hospital stay to outright lethality. C. diff causes cellular damage through the action of two large toxins TcdA and TcdB. Recently, there has been increased effort toward developing antitoxin therapies, rather than antibacterial treatments, in hopes of mitigating the acquisition of drug resistance. To date, no analysis of the recognition mechanism of TcdA or TcdB has been attempted. Here, we use small molecule flexible docking followed by unbiased molecular dynamics to obtain a more detailed perspective on how inhibitory peptides, exemplified by two species HQSPWHH and EGWHAHT function. Using principal component analysis and generalized masked Delaunay analysis, an examination of the conformational space of TcdB in its apo form as well as forms bound to the peptides and UDP-Glucose was performed. Although both species inhibit by binding in the active site, they do so in two very different ways. The simulations show that the conformational space occupied by TcdB bound to the two peptides are quite different and provide valuable insight for the future design of toxin inhibitors and other enzymes that interact with their substrates through conformational capture mechanisms and thus work by the disruption of the protein's intrinsic motions.


Assuntos
Proteínas de Bactérias/química , Toxinas Bacterianas/química , Enterotoxinas/química , Inibidores Enzimáticos/farmacologia , Simulação de Dinâmica Molecular , Sequência de Aminoácidos , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/antagonistas & inibidores , Toxinas Bacterianas/metabolismo , Domínio Catalítico , Enterotoxinas/antagonistas & inibidores , Enterotoxinas/metabolismo , Inibidores Enzimáticos/química , Simulação de Acoplamento Molecular , Dados de Sequência Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Uridina Difosfato Glucose/química , Uridina Difosfato Glucose/farmacologia
4.
ACS Chem Biol ; 5(12): 1097-103, 2010 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-20863124

RESUMO

Clostridium difficile causes severe hospital-acquired antibiotic-associated diarrhea due to the activity of two large protein toxins. Current treatments suffer from a high relapse rate and are generating resistant strains; thus new methods of dealing with these infections that target the virulence factors directly are of interest. Phage display was used to identify peptides that bind to the catalytic domain of C. difficile Toxin A. Library screening and subsequent quantitative binding and inhibition studies showed that several of these peptides are potent inhibitors. Fragment-based computational docking of these peptides elucidated the binding modes within the active site. These antitoxin peptides may serve as potential lead compounds to further engineer peptidomimetic inhibitors of the clostridial toxins.


Assuntos
Antibacterianos/química , Toxinas Botulínicas Tipo A/antagonistas & inibidores , Toxinas Botulínicas/antagonistas & inibidores , Clostridioides difficile/efeitos dos fármacos , Peptídeos/química , Sequência de Aminoácidos , Antibacterianos/farmacologia , Clostridioides difficile/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Modelos Moleculares , Peptídeos/farmacologia , Estrutura Terciária de Proteína
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