Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Genomics ; 98(6): 422-30, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21903159

RESUMEN

Four custom Axiom genotyping arrays were designed for a genome-wide association (GWA) study of 100,000 participants from the Kaiser Permanente Research Program on Genes, Environment and Health. The array optimized for individuals of European race/ethnicity was previously described. Here we detail the development of three additional microarrays optimized for individuals of East Asian, African American, and Latino race/ethnicity. For these arrays, we decreased redundancy of high-performing SNPs to increase SNP capacity. The East Asian array was designed using greedy pairwise SNP selection. However, removing SNPs from the target set based on imputation coverage is more efficient than pairwise tagging. Therefore, we developed a novel hybrid SNP selection method for the African American and Latino arrays utilizing rounds of greedy pairwise SNP selection, followed by removal from the target set of SNPs covered by imputation. The arrays provide excellent genome-wide coverage and are valuable additions for large-scale GWA studies.


Asunto(s)
Pueblo Asiatico/genética , Negro o Afroamericano/genética , Estudio de Asociación del Genoma Completo/métodos , Hispánicos o Latinos/genética , Polimorfismo de Nucleótido Simple , Algoritmos , Asia Oriental , Genoma Humano , Genotipo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Proyectos Piloto , Población Blanca/genética
2.
Genomics ; 98(2): 79-89, 2011 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-21565264

RESUMEN

The success of genome-wide association studies has paralleled the development of efficient genotyping technologies. We describe the development of a next-generation microarray based on the new highly-efficient Affymetrix Axiom genotyping technology that we are using to genotype individuals of European ancestry from the Kaiser Permanente Research Program on Genes, Environment and Health (RPGEH). The array contains 674,517 SNPs, and provides excellent genome-wide as well as gene-based and candidate-SNP coverage. Coverage was calculated using an approach based on imputation and cross validation. Preliminary results for the first 80,301 saliva-derived DNA samples from the RPGEH demonstrate very high quality genotypes, with sample success rates above 94% and over 98% of successful samples having SNP call rates exceeding 98%. At steady state, we have produced 462 million genotypes per week for each Axiom system. The new array provides a valuable addition to the repertoire of tools for large scale genome-wide association studies.


Asunto(s)
Estudio de Asociación del Genoma Completo/métodos , Ensayos Analíticos de Alto Rendimiento , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Polimorfismo de Nucleótido Simple/genética , Población Blanca/genética , Humanos
3.
Arch Biochem Biophys ; 498(1): 13-22, 2010 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-20227382

RESUMEN

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.


Asunto(s)
Dominio Catalítico , ADN (Citosina-5-)-Metiltransferasas/química , ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN/metabolismo , Inhibidores Enzimáticos/farmacología , Eliminación de Secuencia , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Secuencia Conservada , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/genética , ADN Metiltransferasa 3A , Humanos , Nucleótidos de Inosina/química , Nucleótidos de Inosina/farmacología , Cinética , Datos de Secuencia Molecular , Oligonucleótidos/genética , Oligonucleótidos/farmacología , Polímeros/química , Estructura Terciaria de Proteína/genética
4.
Biochemistry ; 48(33): 7807-16, 2009 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-19580326

RESUMEN

Enzymatic sequence-specific DNA modification involves multiple poorly understood intermediates. DNA methyltransferases like M.HhaI initially bind nonspecific DNA and then selectively bind and modify a unique sequence. High-resolution NMR was used to map conformational changes occurring in M.HhaI upon binding nonspecific DNA, a one base pair altered noncognate DNA sequence, and both hemimethylated and unmethylated cognate DNA sequences. Comparisons with previous NMR studies of the apoenzyme and enzyme-cofactor complex provide snapshots of the pathway to sequence-specific complex formation. Dramatic chemical shift perturbations reaching many distal sites within the protein are detected with cognate DNA, while much smaller changes are observed upon nonspecific and noncognate DNA binding. A cooperative rather than stepwise transition from a nonspecific to a cognate complex is revealed. Furthermore, switching from unmethylated to hemimethylated cognate DNA involves detectable protein conformational changes 20-30 A away from the methyl group, indicating high protein sensitivity and plasticity to DNA modification.


Asunto(s)
ADN-Citosina Metilasas/química , Haemophilus/enzimología , Secuencia de Bases , Catálisis , Dominio Catalítico/genética , Cristalografía por Rayos X , Metilación de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , ADN-Citosina Metilasas/genética , Estabilidad de Enzimas/genética , Haemophilus/genética , Unión Proteica/genética , Especificidad por Sustrato/genética
5.
Biochemistry ; 46(24): 7261-8, 2007 Jun 19.
Artículo en Inglés | MEDLINE | ID: mdl-17523600

RESUMEN

The bacterial DNA cytosine methyltransferase M.HhaI sequence-specifically modifies DNA in an S-adenosylmethionine dependent reaction. The enzyme stabilizes the target cytosine (GCGC) into an extrahelical position, with a concomitant large movement of an active site loop involving residues 80-99. We used multidimensional, transverse relaxation-optimized NMR experiments to assign nearly 80% of all residues in the cofactor-bound enzyme form, providing a basis for detailed structural and dynamical characterization. We examined details of the previously unknown effects of the cofactor binding with M.HhaI in solution. Addition of the cofactor results in numerous structural changes throughout the protein, including those decorating the cofactor binding site, and distal residues more than 30 A away. The active site loop is involved in motions both on a picosecond to nanosecond time scale and on a microsecond to millisecond time scale and is not significantly affected by cofactor binding except for a few N-terminal residues. The cofactor also affects residues near the DNA binding cleft, suggesting a role for the cofactor in regulating DNA interactions. The allosteric properties we observed appear to be closely related to the significant amount of dynamics and dynamical changes in response to ligand binding detected in the protein.


Asunto(s)
ADN-Citosina Metilasas/química , ADN-Citosina Metilasas/metabolismo , Regulación Alostérica , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Coenzimas/química , Coenzimas/metabolismo , Cartilla de ADN/genética , ADN-Citosina Metilasas/genética , Ligandos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , S-Adenosilhomocisteína/química , S-Adenosilhomocisteína/metabolismo , Especificidad por Sustrato , Termodinámica
6.
Biochemistry ; 45(51): 15793-806, 2006 Dec 26.
Artículo en Inglés | MEDLINE | ID: mdl-17176102

RESUMEN

The early steps in dioxygen activation by the monooxygenase cytochrome P450cam (CYP101) include binding of O2 to ferrous P450cam to yield the ferric-superoxo form (oxyP450cam) followed by an irreversible, long-range electron transfer from putidaredoxin to reduce the oxyP450cam. The steady state kinetic parameter kcat/Km(O2) has been studied by a variety of probes that indicate a small D2O solvent isotope effect (1.21 +/- 0.08), a very small solvent viscosogen effect, and a 16O/18O isotope effect of 1.0147 +/- 0.0007. This latter value, which can be compared with the 16O/18O equilibrium isotope effect of 1.0048 +/- 0.0003 measured for oxyP450cam formation, is attributed to a primarily rate-limiting outer-sphere electron transfer from the heme iron center as O2 that has prebound to protein approaches the active site cofactor. The electron transfer from putidaredoxin to oxyP450cam was investigated by rapid mixing at 25 degrees C to complement previous lower-temperature measurements. A rate of 390 +/- 23 s-1 (and a near-unity solvent isotope effect) supports the view that the long-range electron transfer from reduced putidaredoxin to oxyP450cam is rapid relative to dissociation of O2 from the enzyme. P450cam represents the first enzymatic reaction of O2 in which both equilibrium and kinetic 16O/18O isotope effects have been measured.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Alcanfor 5-Monooxigenasa/química , Alcanfor 5-Monooxigenasa/metabolismo , Óxido de Deuterio/química , Oxígeno/química , Sitios de Unión , Alcanfor/química , Alcanfor/metabolismo , Catálisis , Óxido de Deuterio/metabolismo , Transporte de Electrón , Ferredoxinas/química , Ferredoxinas/metabolismo , Glucosa/química , Glicerol/química , Cinética , Oxidación-Reducción , Oxígeno/metabolismo , Consumo de Oxígeno , Isótopos de Oxígeno/química , Isótopos de Oxígeno/metabolismo , Protones , Solventes , Sacarosa/química , Viscosidad
7.
Proc Natl Acad Sci U S A ; 102(4): 994-9, 2005 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-15657135

RESUMEN

Molecular dynamics (MD) simulations of HhaI DNA methyltransferase and statistical coupling analysis (SCA) data on the DNA cytosine methyltransferase family were combined to identify residues that are coupled by coevolution and motion. The highest ranking correlated pairs from the data matrix product (SCA.MD) are colocalized and form stabilizing interactions; the anticorrelated pairs are separated on average by 30 A and form a clear focal point centered near the active site. We suggest that these distal anti-correlated pairs are involved in mediating active-site compressions that may be important for catalysis. Mutants that disrupt the implicated interactions support the validity of our combined SCA.MD approach.


Asunto(s)
ADN-Citosina Metilasas/química , Sitios de Unión , Catálisis , ADN-Citosina Metilasas/metabolismo , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad
8.
Biochemistry ; 43(1): 271-81, 2004 Jan 13.
Artículo en Inglés | MEDLINE | ID: mdl-14705955

RESUMEN

Cytochrome P450cam (CYP101) is a prokaryotic monooxygenase that requires two proteins, putidaredoxin reductase (PdR) and putidaredoxin (Pdx), to supply electrons from NADH. This study addresses the mechanism by which electrons are transported from PdR to P450cam through Pdx and used to activate O(2) at the heme of P450cam. It is shown that k(cat)/Km(O2) is independent of the PdR concentration and hyperbolically dependent on Pdx. The phenomenon of saturation of reaction rates with either P450cam or PdR at high ratios of one enzyme to the other is investigated and shown to be consistent with a change in the rate limiting step. Either the reduction of Pdx by PdR (high P450) or the reduction of P450 by Pdx (high PdR) determines the rate. These data support a mechanism where Pdx acts as a shuttle for transport of electrons from PdR to P450cam, effectively ruling out the formation of a kinetically significant PdR/Pdx/P450cam complex.


Asunto(s)
Proteínas Bacterianas/química , Alcanfor 5-Monooxigenasa/química , Oxígeno/química , Catálisis , Citocromos c/química , Transporte de Electrón , Ferredoxinas/química , Cinética , Modelos Químicos , NADH NADPH Oxidorreductasas/química , Oxidación-Reducción , Consumo de Oxígeno , Pseudomonas putida/enzimología , Proteínas Recombinantes/química
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA