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silencing of her2, ccnb1 and pkc genes by sirna results in prolonged retardation of neuroblastoma cell division.deregulation of the expression of the genes that are involved in the control of the cell cycle impairs cellular differentiation and leads to cell death. this process can result in uncontrollable cell proliferation and, subsequently, cancer development. in this study, we examined the effect of the silencing of cancer-related genes by small interfering rnas (sirna) targeted at mrnaof her2, cyclin b1 (ccnb1), and protein kinase c(pkc) on the proliferation of human cancer cells of different origins. ...201122649691
anti-infective potential of hot-spring bacteria.antibiotic resistance currently spans most of the known classes of natural and synthetic antibiotics; limiting our options for treatment of infections and demanding discovery of new classes of antibiotics. much effort is being directed towards developing new antibiotics to overcome this problem. success in getting novel chemical entities from microbial sources depends essentially on novelty of its habitat. the diversity of geographical location decides the type of micro-flora. in the past variou ...201121887055
bypass of a nick by the replisome of bacteriophage t7.dna polymerase and dna helicase are essential components of dna replication. the helicase unwinds duplex dna to provide single-stranded templates for dna synthesis by the dna polymerase. in bacteriophage t7, movement of either the dna helicase or the dna polymerase alone terminates upon encountering a nick in duplex dna. using a minicircular dna, we show that the helicase · polymerase complex can bypass a nick, albeit at reduced efficiency of 7%, on the non-template strand to continue rolling ci ...201121701044
microbial ecology of the dark ocean above, at, and below the seafloor.the majority of life on earth--notably, microbial life--occurs in places that do not receive sunlight, with the habitats of the oceans being the largest of these reservoirs. sunlight penetrates only a few tens to hundreds of meters into the ocean, resulting in large-scale microbial ecosystems that function in the dark. our knowledge of microbial processes in the dark ocean-the aphotic pelagic ocean, sediments, oceanic crust, hydrothermal vents, etc.-has increased substantially in recent decades. ...201121646433
dna polymerases provide a canon of strategies for translesion synthesis past oxidatively generated lesions.deducing the structure of the dna double helix in 1953 implied the mode of its replication: watson-crick (wc) base pairing might instruct an enzyme, now known as the dna polymerase, during the synthesis of a daughter stand complementary to a single strand of the parental double helix. what has become increasingly clear in the last 60 years, however, is that adducted and oxidatively generated dna bases are ubiquitous in physiological dna, and all organisms conserve multiple dna polymerases specia ...201121482102
role of iron deficiency anemia in the propagation of beta thalssemia gene.the diagnostic criterion for beta thalassemia trait (btt) is elevated hb-a(2) levels. iron deficiency anemia (ida) reduces the synthesis of hb-a(2), resulting in reduced hb-a(2) levels, so patients with co-pathological conditions btt with ida, may have a normal level of hb-a(2). many socio-economic factors like unawareness, poor diagnostic facilities, and cost of molecular diagnosis (for screening purposes) result in interpretation of these subjects as normal.201121461303
mechanism of bacterial transcription initiation: rna polymerase - promoter binding, isomerization to initiation-competent open complexes, and initiation of rna synthesis.initiation of rna synthesis from dna templates by rna polymerase (rnap) is a multi-step process, in which initial recognition of promoter dna by rnap triggers a series of conformational changes in both rnap and promoter dna. the bacterial rnap functions as a molecular isomerization machine, using binding free energy to remodel the initial recognition complex, placing downstream duplex dna in the active site cleft and then separating the nontemplate and template strands in the region surrounding ...201121371479
molecular mechanism of co-translational protein targeting by the signal recognition particle.the signal recognition particle (srp) is a key component of the cellular machinery that couples the ongoing synthesis of proteins to their proper localization, and has often served as a paradigm for understanding the molecular basis of protein localization within the cell. the srp pathway exemplifies several key molecular events required for protein targeting to cellular membranes: the specific recognition of signal sequences on cargo proteins, the efficient delivery of cargo to the target membr ...201121291501
x-ray crystal structures elucidate the nucleotidyl transfer reaction of transcript initiation using two nucleotides.we have determined the x-ray crystal structures of the pre- and postcatalytic forms of the initiation complex of bacteriophage n4 rna polymerase that provide the complete set of atomic images depicting the process of transcript initiation by a single-subunit rna polymerase. as observed during t7 rna polymerase transcript elongation, substrate loading for the initiation process also drives a conformational change of the o-helix, but only the correct base pairing between the +2 substrate and dna b ...201121321236
dna polymerases and cancer.there are 15 different dna polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of dna damage. new evidence is emerging for lesion-specific and tissue-specific functions of dna polymerases. many point mutations that occur in cancer cells arise from the error-generating activities of dna polymerases. however, the ability of some of these enzymes to bypass dna damage may actually defend against chromosome instability in cells, and at least one dn ...201121258395
archaeal rna polymerase and transcription regulation.to elucidate the mechanism of transcription by cellular rna polymerases (rnaps), high-resolution x-ray crystal structures together with structure-guided biochemical, biophysical, and genetics studies are essential. the recently solved x-ray crystal structures of archaeal rnap allow a structural comparison of the transcription machinery among all three domains of life. the archaea were once thought of closely related to bacteria, but they are now considered to be more closely related to the eukar ...201121250781
use of base modifications in primers and amplicons to improve nucleic acids detection in the real-time snake polymerase chain reaction.the addition of relatively short flap sequence at the 5'-end of one of the polymerase chain reaction (pcr) primers considerably improves performance of real-time assays based on 5'-nuclease activity. this new technology, called snake, was shown to supersede the conventional methods like taqman, molecular beacons, and scorpions in the signal productivity and discrimination of target polymorphic variations as small as single nucleotides. the present article describes a number of reaction condition ...201121050073
unlocking the sugar "steric gate" of dna polymerases.to maintain genomic stability, ribonucleotide incorporation during dna synthesis is controlled predominantly at the dna polymerase level. a steric clash between the 2'-hydroxyl of an incoming ribonucleotide and a bulky active site residue, known as the "steric gate", establishes an effective mechanism for most dna polymerases to selectively insert deoxyribonucleotides. recent kinetic, structural, and in vivo studies have illuminated novel features about ribonucleotide exclusion and the mechanist ...201121226515
controlled interplay between trigger loop and gre factor in the rna polymerase active centre.the highly processive transcription by multi-subunit rna polymerases (rnap) can be interrupted by misincorporation or backtracking events that may stall transcription or lead to erroneous transcripts. backtracked/misincorporated complexes can be resolved via hydrolysis of the transcript. here, we show that, in response to misincorporation and/or backtracking, the catalytic domain of rnap active centre, the trigger loop (tl), is substituted by transcription factor gre. this substitution turns off ...201121266474
association of tumor necrosis factor β genetic polymorphism and sepsis susceptibility.the association of the tumor necrosis factor β (tnf-β) nco1 genetic polymorphism with susceptibility to sepsis was evaluated in 60 consecutive patients diagnosed with sepsis and in 148 healthy blood donors. genomic dna was extracted from peripheral blood cells and a 782 base-pair fragment of the tnf-β gene was amplified by pcr. the pcr products were subjected to nco1 restriction digestion and analysed by restriction fragment length polymorphism analysis. tumor necrosis factor α (tnf-α) and the c ...201122977509
mutations in the external loops of bk virus vp1 and urine viral load in renal transplant recipients.polyomavirus-associated nephropathy (pvan) is a major complication that occurs after renal transplantation and is induced by reactivation of the human polyomavirus bk (bkv). the structure of the viral capsid protein 1 (vp1) is characterized by the presence of external loops, bc, de, ef, gh, and hi, which are involved in receptor binding. the pathogenesis of pvan is not well understood, but viral risk factors are thought to play a crucial role in the onset of this pathology. in an attempt to bett ...201019780025
finding chimeras: a bioinformatics strategy for identification of cross-linked peptides.chemical cross-linking, followed by identification of the cross-linked residues, is a powerful approach to probe the topologies and interacting surfaces of protein assemblies. in this work, we demonstrate a new bioinformatics approach using multiple program modules within the software package "protein prospector" that greatly facilitates the discovery of cross-linked peptides in chemical cross-linking studies. examples are given for how this approach has been used for defining interfaces in hete ...201019809093
finding chimeras: a bioinformatics strategy for identification of cross-linked peptides.chemical cross-linking, followed by identification of the cross-linked residues, is a powerful approach to probe the topologies and interacting surfaces of protein assemblies. in this work, we demonstrate a new bioinformatics approach using multiple program modules within the software package "protein prospector" that greatly facilitates the discovery of cross-linked peptides in chemical cross-linking studies. examples are given for how this approach has been used for defining interfaces in hete ...201019809093
proteomics-based refinement of deinococcus deserti genome annotation reveals an unwonted use of non-canonical translation initiation codons.deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. to achieve a comprehensive and accurate annotation of the deinococcus deserti genome, we performed an n terminus-oriented characterization of its proteome. for this, we used a labeling reagent, n-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein n termini. the larg ...201019875382
proteomics-based refinement of deinococcus deserti genome annotation reveals an unwonted use of non-canonical translation initiation codons.deinococcaceae are a family of extremely radiation-tolerant bacteria that are currently subjected to numerous studies aimed at understanding the molecular mechanisms for such radiotolerance. to achieve a comprehensive and accurate annotation of the deinococcus deserti genome, we performed an n terminus-oriented characterization of its proteome. for this, we used a labeling reagent, n-tris(2,4,6-trimethoxyphenyl)phosphonium acetyl succinimide, to selectively derivatize protein n termini. the larg ...201019875382
molecular evolution of multisubunit rna polymerases: structural analysis.comprehensive multiple sequence alignments of the multisubunit dna-dependent rna polymerase (rnap) large subunits, including the bacterial beta and beta' subunits and their homologs from archaebacterial rnaps, eukaryotic rnaps i-iii, nuclear-cytoplasmic large double-stranded dna virus rnaps, and plant plastid rnaps, were created [lane, w. j. and darst, s. a. (2009). molecular evolution of multisubunit rna polymerases: sequence analysis. in press]. the alignments were used to delineate sequence r ...201019895816
molecular evolution of multisubunit rna polymerases: structural analysis.comprehensive multiple sequence alignments of the multisubunit dna-dependent rna polymerase (rnap) large subunits, including the bacterial beta and beta' subunits and their homologs from archaebacterial rnaps, eukaryotic rnaps i-iii, nuclear-cytoplasmic large double-stranded dna virus rnaps, and plant plastid rnaps, were created [lane, w. j. and darst, s. a. (2009). molecular evolution of multisubunit rna polymerases: sequence analysis. in press]. the alignments were used to delineate sequence r ...201019895816
stereochemical mechanisms of trna methyltransferases.methylation of trna on the four canonical bases adds structural complexity to the molecule, and improves decoding specificity and efficiency. while many trna methylases are known, detailed insight into the catalytic mechanism is only available in a few cases. of interest among all trna methylases is the structural basis for nucleotide selection, by which the specificity is limited to a single site, or broadened to multiple sites. general themes in catalysis include the basis for rate acceleratio ...201019944101
an adenylyl cyclase signaling pathway predicts direct dopaminergic input to vestibular hair cells.adenylyl cyclase (ac) signaling pathways have been identified in a model hair cell preparation from the trout saccule, for which the hair cell is the only intact cell type. the use of degenerate primers targeting cdna sequence conserved across ac isoforms, and reverse transcription-polymerase chain reaction (rt-pcr), coupled with cloning of amplification products, indicated expression of ac9, ac7 and ac5/6, with cloning efficiencies of 11:5:2. ac9 and ac5/6 are inhibited by ca(2+), the former in ...201020883745
rational design, synthesis, and evaluation of new selective inhibitors of microbial class ii (zinc dependent) fructose bis-phosphate aldolases.we report the synthesis and biochemical evaluation of several selective inhibitors of class ii (zinc dependent) fructose bis-phosphate aldolases (fba). the products were designed as transition-state analogues of the catalyzed reaction, structurally related to the substrate fructose bis-phosphate (or sedoheptulose bis-phosphate) and based on an n-substituted hydroxamic acid, as a chelator of the zinc ion present in active site. the compounds synthesized were tested on class ii fbas from various p ...201020929256
the core-independent promoter-specific interaction of primary sigma factor.previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. most σ functions were studied on the basis of this tenet. here, we provide in vitro evidence demonstrating that the intact bacillus subtilis primary sigma, σ(a), by itself, is able to interact specifically with promoter deoxyribonucleic acid (dna), albeit with low sequence selectivity. the core-independent promoter-specific interaction of t ...201020935043
the core-independent promoter-specific interaction of primary sigma factor.previous studies have led to a model in which the promoter-specific recognition of prokaryotic transcription initiation factor, sigma (σ), is core dependent. most σ functions were studied on the basis of this tenet. here, we provide in vitro evidence demonstrating that the intact bacillus subtilis primary sigma, σ(a), by itself, is able to interact specifically with promoter deoxyribonucleic acid (dna), albeit with low sequence selectivity. the core-independent promoter-specific interaction of t ...201020935043
reversal of a mutator activity by a nearby fidelity-neutral substitution in the rb69 dna polymerase binding pocket.phage rb69 b-family dna polymerase is responsible for the overall high fidelity of rb69 dna synthesis. fidelity is compromised when conserved tyr567, one of the residues that form the nascent polymerase base-pair binding pocket, is replaced by alanine. the y567a mutator mutant has an enlarged binding pocket and can incorporate and extend mispairs efficiently. ser565 is a nearby conserved residue that also contributes to the binding pocket, but a s565g replacement has only a small impact on dna r ...201020950625
structures of a key interaction protein from the trypanosoma brucei editosome in complex with single domain antibodies.several major global diseases are caused by single-cell parasites called trypanosomatids. these organisms exhibit many unusual features including a unique and essential u-insertion/deletion rna editing process in their single mitochondrion. many key rna editing steps occur in ∼20s editosomes, which have a core of 12 proteins. among these, the "interaction protein" krepa6 performs a central role in maintaining the integrity of the editosome core and also binds to ssrna. the use of llama single do ...201020969962
structures of a key interaction protein from the trypanosoma brucei editosome in complex with single domain antibodies.several major global diseases are caused by single-cell parasites called trypanosomatids. these organisms exhibit many unusual features including a unique and essential u-insertion/deletion rna editing process in their single mitochondrion. many key rna editing steps occur in ∼20s editosomes, which have a core of 12 proteins. among these, the "interaction protein" krepa6 performs a central role in maintaining the integrity of the editosome core and also binds to ssrna. the use of llama single do ...201020969962
site-directed mutagenesis of the χ subunit of dna polymerase iii and single-stranded dna-binding protein of e. coli reveals key residues for their interaction.during dna replication in escherichia coli, single-stranded dna-binding protein (ssb) protects single-stranded dna from nuclease action and hairpin formation. it is known that the highly conserved c-terminus of ssb contacts the χ subunit of dna polymerase iii. however, there only exists a theoretical model in which the 11 c-terminal amino acids of ssb have been docked onto the surface of χ. in order to refine this model of ssb/χ interaction, we exchanged amino acids in χ and ssb by site-directed ...201020972214
site-directed mutagenesis of the χ subunit of dna polymerase iii and single-stranded dna-binding protein of e. coli reveals key residues for their interaction.during dna replication in escherichia coli, single-stranded dna-binding protein (ssb) protects single-stranded dna from nuclease action and hairpin formation. it is known that the highly conserved c-terminus of ssb contacts the χ subunit of dna polymerase iii. however, there only exists a theoretical model in which the 11 c-terminal amino acids of ssb have been docked onto the surface of χ. in order to refine this model of ssb/χ interaction, we exchanged amino acids in χ and ssb by site-directed ...201020972214
omnipotent role of archaeal elongation factor 1 alpha (ef1α in translational elongation and termination, and quality control of protein synthesis.the molecular mechanisms of translation termination and mrna surveillance in archaea remain unclear. in eukaryotes, erf3 and hbs1, which are homologous to the trna carrier gtpase ef1α, respectively bind erf1 and pelota to decipher stop codons or to facilitate mrna surveillance. however, genome-wide searches of archaea have failed to detect any orthologs to both gtpases. here, we report the crystal structure of arf1 from an archaeon, aeropyrum pernix, and present strong evidence that the authenti ...201020974926
molecular mechanisms of the whole dna repair system: a comparison of bacterial and eukaryotic systems.dna is subjected to many endogenous and exogenous damages. all organisms have developed a complex network of dna repair mechanisms. a variety of different dna repair pathways have been reported: direct reversal, base excision repair, nucleotide excision repair, mismatch repair, and recombination repair pathways. recent studies of the fundamental mechanisms for dna repair processes have revealed a complexity beyond that initially expected, with inter- and intrapathway complementation as well as f ...201020981145
transcription of the t4 late genes.this article reviews the current state of understanding of the regulated transcription of the bacteriophage t4 late genes, with a focus on the underlying biochemical mechanisms, which turn out to be unique to the t4-related family of phages or significantly different from other bacterial systems. the activator of t4 late transcription is the gene 45 protein (gp45), the sliding clamp of the t4 replisome. gp45 becomes topologically linked to dna through the action of its clamp-loader, but it is no ...201021029432
temporal regulation of gene expression of the thermus thermophilus bacteriophage p23-45.regulation of gene expression during infection of the thermophilic bacterium thermus thermophilus hb8 with the bacteriophage p23-45 was investigated. macroarray analysis revealed host transcription shut-off and identified three temporal classes of phage genes; early, middle and late. primer extension experiments revealed that the 5' ends of p23-45 early transcripts are preceded by a common sequence motif that likely defines early viral promoters. t. thermophilus hb8 rna polymerase (rnap) recogni ...201021050864
temporal regulation of gene expression of the thermus thermophilus bacteriophage p23-45.regulation of gene expression during infection of the thermophilic bacterium thermus thermophilus hb8 with the bacteriophage p23-45 was investigated. macroarray analysis revealed host transcription shut-off and identified three temporal classes of phage genes; early, middle and late. primer extension experiments revealed that the 5' ends of p23-45 early transcripts are preceded by a common sequence motif that likely defines early viral promoters. t. thermophilus hb8 rna polymerase (rnap) recogni ...201021050864
structural basis for error-free replication of oxidatively damaged dna by yeast dna polymerase η.7,8-dihydro-8-oxoguanine (8-oxog) adducts are formed frequently by the attack of oxygen-free radicals on dna. they are among the most mutagenic lesions in cells because of their dual coding potential, where, in addition to normal base-pairing of 8-oxog(anti) with dctp, 8-oxog in the syn conformation can base pair with datp, causing g to t transversions. we provide here for the first time a structural basis for the error-free replication of 8-oxog lesions by yeast dna polymerase η (polη). we show ...201021070945
trypanosoma cruzi msh2: functional analyses on different parasite strains provide evidences for a role on the oxidative stress response.components of the dna mismatch repair (mmr) pathway are major players in processes known to generate genetic diversity, such as mutagenesis and dna recombination. trypanosoma cruzi, the protozoan parasite that causes chagas disease has a highly heterogeneous population, composed of a pool of strains with distinct characteristics. studies with a number of molecular markers identified up to six groups in the t. cruzi population, which showed distinct levels of genetic variability. to investigate t ...201021073906
structural basis for the synthesis of nucleobase modified dna by thermus aquaticus dna polymerase.numerous 2'-deoxynucleoside triphosphates (dntps) that are functionalized with spacious modifications such as dyes and affinity tags like biotin are substrates for dna polymerases. they are widely employed in many cutting-edge technologies like advanced dna sequencing approaches, microarrays, and single molecule techniques. modifications attached to the nucleobase are accepted by many dna polymerases, and thus, dntps bearing nucleobase modifications are predominantly employed. when pyrimidines a ...201021123743
turning science into health solutions: kemri's challenges as kenya's health product pathfinder.abstract : background : a traditional pathway for developing new health products begins with public research institutes generating new knowledge, and ends with the private sector translating this knowledge into new ventures. but while public research institutes are key drivers of basic research in sub-saharan africa, the private sector is inadequately prepared to commercialize ideas that emerge from these institutes, resulting in these institutes taking on the role of product development themsel ...201021144070
structural basis for the oxidation of protein-bound sulfur by the sulfur cycle molybdohemo-enzyme sulfane dehydrogenase soxcd.the sulfur cycle enzyme sulfane dehydrogenase soxcd is an essential component of the sulfur oxidation (sox) enzyme system of paracoccus pantotrophus. soxcd catalyzes a six-electron oxidation reaction within the sox cycle. soxcd is an α(2)β(2) heterotetrameric complex of the molybdenum cofactor-containing soxc protein and the diheme c-type cytochrome soxd with the heme domains d(1) and d(2). soxcd(1) misses the heme-2 domain d(2) and is catalytically as active as soxcd. the crystal structure of s ...201021147779
structural basis for the oxidation of protein-bound sulfur by the sulfur cycle molybdohemo-enzyme sulfane dehydrogenase soxcd.the sulfur cycle enzyme sulfane dehydrogenase soxcd is an essential component of the sulfur oxidation (sox) enzyme system of paracoccus pantotrophus. soxcd catalyzes a six-electron oxidation reaction within the sox cycle. soxcd is an α(2)β(2) heterotetrameric complex of the molybdenum cofactor-containing soxc protein and the diheme c-type cytochrome soxd with the heme domains d(1) and d(2). soxcd(1) misses the heme-2 domain d(2) and is catalytically as active as soxcd. the crystal structure of s ...201021147779
cryo-em structure of the e. coli translating ribosome in complex with srp and its receptor.we report the 'early' conformation of the escherichia coli signal recognition particle (srp) and its receptor ftsy bound to the translating ribosome, as determined by cryo-em. ftsy binds to the tetraloop of the srp rna, whereas the ng domains of the srp protein and ftsy interact weakly in this conformation. our results suggest that optimal positioning of the srp rna tetraloop and the ffh ng domain leads to ftsy recruitment.201021151118
cryo-em structure of the e. coli translating ribosome in complex with srp and its receptor.we report the 'early' conformation of the escherichia coli signal recognition particle (srp) and its receptor ftsy bound to the translating ribosome, as determined by cryo-em. ftsy binds to the tetraloop of the srp rna, whereas the ng domains of the srp protein and ftsy interact weakly in this conformation. our results suggest that optimal positioning of the srp rna tetraloop and the ffh ng domain leads to ftsy recruitment.201021151118
effect of n2-guanyl modifications on early steps in catalysis of polymerization by sulfolobus solfataricus p2 dna polymerase dpo4 t239w.translesion dna polymerases are more efficient at bypass of many dna adducts than replicative polymerases. previous work with the translesion polymerase sulfolobus solfataricus dpo4 showed a decrease in catalytic efficiency during bypass of bulky n(2)-alkyl guanine (g) adducts with n(2)-isobutylg showing the largest effect, decreasing approximately 120-fold relative to unmodified deoxyguanosine (zhang, h., eoff, r. l., egli, m., guengerich, f. p. versatility of y-family sulfolobus solfataricus d ...201019969000
effect of n2-guanyl modifications on early steps in catalysis of polymerization by sulfolobus solfataricus p2 dna polymerase dpo4 t239w.translesion dna polymerases are more efficient at bypass of many dna adducts than replicative polymerases. previous work with the translesion polymerase sulfolobus solfataricus dpo4 showed a decrease in catalytic efficiency during bypass of bulky n(2)-alkyl guanine (g) adducts with n(2)-isobutylg showing the largest effect, decreasing approximately 120-fold relative to unmodified deoxyguanosine (zhang, h., eoff, r. l., egli, m., guengerich, f. p. versatility of y-family sulfolobus solfataricus d ...201019969000
new approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of forster resonance energy transfer probes in 5'-nuclease assays.the article describes a new technology for real-time polymerase chain reaction (pcr) detection of nucleic acids. similar to taqman, this new method, named snake, utilizes the 5'-nuclease activity of thermus aquaticus (taq) dna polymerase that cleaves dual-labeled förster resonance energy transfer (fret) probes and generates a fluorescent signal during pcr. however, the mechanism of the probe cleavage in snake is different. in this assay, pcr amplicons fold into stem-loop secondary structures. hy ...201019969535
new approach to real-time nucleic acids detection: folding polymerase chain reaction amplicons into a secondary structure to improve cleavage of forster resonance energy transfer probes in 5'-nuclease assays.the article describes a new technology for real-time polymerase chain reaction (pcr) detection of nucleic acids. similar to taqman, this new method, named snake, utilizes the 5'-nuclease activity of thermus aquaticus (taq) dna polymerase that cleaves dual-labeled förster resonance energy transfer (fret) probes and generates a fluorescent signal during pcr. however, the mechanism of the probe cleavage in snake is different. in this assay, pcr amplicons fold into stem-loop secondary structures. hy ...201019969535
mobilization studies in complement-deficient mice reveal that optimal amd3100 mobilization of hematopoietic stem cells depends on complement cascade activation by amd3100-stimulated granulocytes.we reported that complement cascade (cc) becomes activated in bone marrow (bm) during mobilization of hematopoietic stem/progenitor cells (hspcs) induced by granulocyte colony-stimulating factor (g-csf) and c5 cleavage has an important function in optimal egress of hspcs. in this work, we explored whether cc is involved in mobilization of hspcs induced by the cxcr4 antagonist, amd3100. to address this question, we performed mobilization studies in mice that display a defect in the activation of ...201020033053
mobilization studies in complement-deficient mice reveal that optimal amd3100 mobilization of hematopoietic stem cells depends on complement cascade activation by amd3100-stimulated granulocytes.we reported that complement cascade (cc) becomes activated in bone marrow (bm) during mobilization of hematopoietic stem/progenitor cells (hspcs) induced by granulocyte colony-stimulating factor (g-csf) and c5 cleavage has an important function in optimal egress of hspcs. in this work, we explored whether cc is involved in mobilization of hspcs induced by the cxcr4 antagonist, amd3100. to address this question, we performed mobilization studies in mice that display a defect in the activation of ...201020033053
characterization of nucleotide misincorporation patterns in the iceman's mitochondrial dna.the degradation of dna represents one of the main issues in the genetic analysis of archeological specimens. in the recent years, a particular kind of post-mortem dna modification giving rise to nucleotide misincorporation ("miscoding lesions") has been the object of extensive investigations.201020072618
saccharomyces cerevisiae msh2-msh6 dna binding kinetics reveal a mechanism of targeting sites for dna mismatch repair.the dna mismatch repair system (mmr) identifies replication errors and damaged bases in dna and functions to preserve genomic integrity. muts performs the task of locating mismatched base pairs, loops and lesions and initiating mmr, and the fundamental question of how this protein targets specific sites in dna is unresolved. to address this question, we examined the interactions between saccharomyces cerevisiae msh2-msh6, a eukaryotic muts homolog, and dna in real time. the reaction kinetics rev ...201020080735
saccharomyces cerevisiae msh2-msh6 dna binding kinetics reveal a mechanism of targeting sites for dna mismatch repair.the dna mismatch repair system (mmr) identifies replication errors and damaged bases in dna and functions to preserve genomic integrity. muts performs the task of locating mismatched base pairs, loops and lesions and initiating mmr, and the fundamental question of how this protein targets specific sites in dna is unresolved. to address this question, we examined the interactions between saccharomyces cerevisiae msh2-msh6, a eukaryotic muts homolog, and dna in real time. the reaction kinetics rev ...201020080735
a mechanistic view of human mitochondrial dna polymerase gamma: providing insight into drug toxicity and mitochondrial disease.mitochondrial dna polymerase gamma (pol gamma) is the sole polymerase responsible for replication of the mitochondrial genome. the study of human pol gamma is of key importance to clinically relevant issues such as nucleoside analog toxicity and mitochondrial disorders such as progressive external ophthalmoplegia. the development of a recombinant form of the human pol gamma holoenzyme provided an essential tool in understanding the mechanism of these clinically relevant phenomena using kinetic m ...201020083238
evaluation of localized and systemic immune responses in cutaneous leishmaniasis caused by leishmania tropica: interleukin-8, monocyte chemotactic protein-1 and nitric oxide are major regulatory factors.we have established leishmania tropica as the causative agent of cutaneous leishmaniasis (cl) in the region of india where the disease is endemic. the association between localized and circulating levels of immune-determinants in cl patients was evaluated. reverse transcription-polymerase chain reaction analysis revealed up-regulation of interferon-gamma (ifn-gamma), interleukin (il)-1beta, il-8, tumour necrosis factor-alpha (tnf-alpha), il-10 and il-4 in dermal lesions at the pretreatment stage ...201020102417
primase directs the release of dnac from dnab.an aaa+ atpase, dnac, delivers dnab helicase at the e. coli chromosomal origin by a poorly understood process. this report shows that mutant proteins bearing alanine substitutions for two conserved arginines in a motif named box vii are defective in dna replication, but this deficiency does not arise from impaired interactions with atp, dnab, or single-stranded dna. despite their ability to deliver dnab to the chromosomal origin to form the prepriming complex, this intermediate is inactive. quan ...201020129058
t7 phage protein gp2 inhibits the escherichia coli rna polymerase by antagonizing stable dna strand separation near the transcription start site.infection of escherichia coli by the t7 phage leads to rapid and selective inhibition of the host rna polymerase (rnap)--a multi-subunit enzyme responsible for gene transcription--by a small ( approximately 7 kda) phage-encoded protein called gp2. gp2 is also a potent inhibitor of e. coli rnap in vitro. here we describe the first atomic resolution structure of gp2, which reveals a distinct run of surface-exposed negatively charged amino acid residues on one side of the molecule. our comprehensiv ...201020133868
muts and mutl are dispensable for maintenance of the genomic mutation rate in the halophilic archaeon halobacterium salinarum nrc-1.the genome of the halophilic archaeon halobacterium salinarum nrc-1 encodes for homologs of muts and mutl, which are key proteins of a dna mismatch repair pathway conserved in bacteria and eukarya. mismatch repair is essential for retaining the fidelity of genetic information and defects in this pathway result in the deleterious accumulation of mutations and in hereditary diseases in humans.201020140215
mutagenic potential of dna glycation: miscoding by (r)- and (s)-n2-(1-carboxyethyl)-2'-deoxyguanosine.elevated circulating glucose resulting from complications of obesity and metabolic disease can result in the accumulation of advanced glycation end products (ages) of proteins, lipids, and dna. the formation of dna-ages assumes particular importance as these adducts may contribute to genetic instability and elevated cancer risk associated with metabolic disease. the principal dna-age, n(2)-(1-carboxyethyl)-2'-deoxyguanosine (cedg), is formed as a mixture of r and s isomers at both the polymer an ...201020143879
evolutionary conservation of residues in vertebrate dna polymerase n conferring low fidelity and bypass activity.poln is a nuclear a-family dna polymerase encoded in vertebrate genomes. poln has unusual fidelity and dna lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of t for template g and accurate translesion synthesis past a 5s-thymine glycol (5s-tg). we searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol i-type dna polymerases. a lys residue (679 in human poln) of particular interest was identifie ...201020144948
characterization of endott, a novel single-stranded dna-specific endonuclease from thermoanaerobacter tengcongensis.endott encoded by tte0829 of thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) dna in vitro as well as binding dsdna. in the presence of a low concentration of nacl, endott cleaved ss regions of damaged dsdna efficiently but did not cleave dna that was entirely ss or ds. at high concentrations of nacl or mgcl(2) or atp, there was also specific cleavage of ssdna. this suggested a preference for ss/ds junctions to stimulate cleavage of the dna ...201020172959
involvement of the beta subunit of rna polymerase in resistance to streptolydigin and streptovaricin in the producer organisms streptomyces lydicus and streptomyces spectabilis.streptomyces lydicus nrrl2433 and s. spectabilis nrrl2494 produce two inhibitors of bacterial rna polymerase: the 3-acyltetramic acid streptolydigin and the naphthalenic ansamycin streptovaricin, respectively. both strains are highly resistant to their own antibiotics. independent expression of the s. lydicus and s. spectabilis rpob and rpoc genes, encoding the beta- and beta'-subunits of rna polymerase, respectively, in s. albus showed that resistance is mediated by rpob, with no effect of rpoc ...201020176899
probing dna- and atp-mediated conformational changes in the muts family of mispair recognition proteins using deuterium exchange mass spectrometry.we have performed deuterium exchange mass spectrometry (dxms) to probe the conformational changes that the bacterial muts homodimer and the homologous eukaryotic heterodimer msh2-msh6 undergo when binding to atp or dna. the dxms data support the view that high affinity binding to mispair-containing dna and low affinity binding to fully base-paired dna both involve forming rings by muts protein family dimers around the dna; however, mispair binding protects additional regions from deuterium excha ...201020181951
recognition of a signal peptide by the signal recognition particle.targeting of proteins to appropriate subcellular compartments is a crucial process in all living cells. secretory and membrane proteins usually contain an amino-terminal signal peptide, which is recognized by the signal recognition particle (srp) when nascent polypeptide chains emerge from the ribosome. the srp-ribosome nascent chain complex is then targeted through its gtp-dependent interaction with srp receptor to the protein-conducting channel on endoplasmic reticulum membrane in eukaryotes o ...201020364120
redox sensing by a rex-family repressor is involved in the regulation of anaerobic gene expression in staphylococcus aureus.an alignment of upstream regions of anaerobically induced genes in staphylococcus aureus revealed the presence of an inverted repeat, corresponding to rex binding sites in streptomyces coelicolor. gel shift experiments of selected upstream regions demonstrated that the redox-sensing regulator rex of s. aureus binds to this inverted repeat. the binding sequence--ttgtgaaw(4)ttcacaa--is highly conserved in s. aureus. rex binding to this sequence leads to the repression of genes located downstream. ...201020374494
structural aspects of drug resistance and inhibition of hiv-1 reverse transcriptase.hiv-1 reverse transcriptase (hiv-1 rt) has been the target of numerous approved anti-aids drugs that are key components of highly active anti-retroviral therapies (haart). it remains the target of extensive structural studies that continue unabated for almost twenty years. the crystal structures of wild-type or drug-resistant mutant hiv rts in the unliganded form or in complex with substrates and/or drugs have offered valuable glimpses into the enzyme's folding and its interactions with dna and ...201020376302
transient tether between the srp rna and srp receptor ensures efficient cargo delivery during cotranslational protein targeting.kinetic control of macromolecular interactions plays key roles in biological regulation. an example of such control occurs in cotranslational protein targeting by the signal recognition particle (srp), during which the srp rna and the cargo both accelerate complex assembly between the srp and srp receptor ftsy 10(2)-fold. the molecular mechanism underlying these rate accelerations was unclear. here we show that a highly conserved basic residue, lys399, on the lateral surface of ftsy provides a n ...201020385832
pyrosequencing-based comparative genome analysis of the nosocomial pathogen enterococcus faecium and identification of a large transferable pathogenicity island.the gram-positive bacterium enterococcus faecium is an important cause of nosocomial infections in immunocompromized patients.201020398277
a unique group of virus-related, genome-integrating elements found solely in the bacterial family thermaceae and the archaeal family halobacteriaceae.viruses sh1 and p23-77, infecting archaeal haloarcula species and bacterial thermus species, respectively, were recently designated to form a novel viral lineage. in this study, the lineage is expanded to archaeal halomicrobium and bacterial meiothermus species by analysis of five genome-integrated elements that share the core genes with these viruses.201020400546
replication through an abasic dna lesion: structural basis for adenine selectivity.abasic sites represent the most frequent dna lesions in the genome that have high mutagenic potential and lead to mutations commonly found in human cancers. although these lesions are devoid of the genetic information, adenine is most efficiently inserted when abasic sites are bypassed by dna polymerases, a phenomenon termed a-rule. in this study, we present x-ray structures of a dna polymerase caught while incorporating a nucleotide opposite an abasic site. we found that a functionally importan ...201020400942
two novel families of plasmids from hyperthermophilic archaea encoding new families of replication proteins.thermococcales (phylum euryarchaeota) are model organisms for physiological and molecular studies of hyperthermophiles. here we describe three new plasmids from thermococcales that could provide new tools and model systems for genetic and molecular studies in archaea. the plasmids ptn2 from thermococcus nautilus sp. 30-1 and pp12-1 from pyrococcus sp. 12-1 belong to the same family. they have similar size (approximately 12 kb) and share six genes, including homologues of genes encoded by the vir ...201020403814
functional studies and homology modeling of msh2-msh3 predict that mispair recognition involves dna bending and strand separation.the msh2-msh3 heterodimer recognizes various dna mispairs, including loops of dna ranging from 1 to 14 nucleotides and some base-base mispairs. homology modeling of the mispair-binding domain (mbd) of msh3 using the related msh6 mbd revealed that mismatch recognition must be different, even though the mbd folds must be similar. model-based point mutation alleles of saccharomyces cerevisiae msh3 designed to disrupt mispair recognition fell into two classes. one class caused defects in repair of b ...201020421420
solution structure of rv2377c-founding member of the mbth-like protein family.the mycobacterium tuberculosis protein rv2377c (71 residues, mw=8.4kda) has been characterized using nuclear magnetic resonance (nmr) and circular dichroism (cd) spectroscopy. rv2377c was the first identified member of the mbth-like family of proteins. mbth-like proteins have been implicated in siderophore biosynthesis, however, their precise biochemical function remain unknown. size exclusion chromatography and nmr spectroscopy show that rv2377c is a monomer in solution. circular dichroism spec ...201020434955
thrombin regulates the metastatic potential of human rhabdomyosarcoma cells: distinct role of par1 and par3 signaling.we observed that human rhabdomyosarcoma (rms) cells highly express a tissue factor that promotes thrombin formation, which indirectly and directly affects rms progression. first, we found that thrombin activates platelets to generate microvesicles (pmv), which transfer to rms cells' alpha2beta3 integrin and increase their adhesiveness to endothelial cells. accordingly, rms cells covered with pmvs showed higher metastatic potential after i.v. injection into immunodeficient mice. furthermore, pmvs ...201020442298
testing computational prediction of missense mutation phenotypes: functional characterization of 204 mutations of human cystathionine beta synthase.predicting the phenotypes of missense mutations uncovered by large-scale sequencing projects is an important goal in computational biology. high-confidence predictions can be an aid in focusing experimental and association studies on those mutations most likely to be associated with causative relationships between mutation and disease. as an aid in developing these methods further, we have derived a set of random mutations of the enzymatic domains of human cystathionine beta synthase. this enzym ...201020455263
multiple roles of the rna polymerase {beta}' sw2 region in transcription initiation, promoter escape, and rna elongation.interactions of rna polymerase (rnap) with nucleic acids must be tightly controlled to ensure precise and processive rna synthesis. the rnap β'-subunit switch-2 (sw2) region is part of a protein network that connects the clamp domain with the rnap body and mediates opening and closing of the active center cleft. sw2 interacts with the template dna near the rnap active center and is a target for antibiotics that block dna melting during initiation. here, we show that substitutions of a conserved ...201020457751
mass spectrometry defines the stoichiometry of ribosomal stalk complexes across the phylogenetic tree.the ribosomal stalk complex plays a crucial role in delivering translation factors to the catalytic site of the ribosome. it has a very similar architecture in all cells, although the protein components in bacteria are unrelated to those in archaea and eukaryotes. here we used mass spectrometry to investigate ribosomal stalk complexes from bacteria, eukaryotes, and archaea in situ on the ribosome. specifically we targeted ribosomes with different optimal growth temperatures. our results showed t ...201020467040
discrimination of thermophilic and mesophilic proteins.there is a considerable literature on the source of the thermostability of proteins from thermophilic organisms. understanding the mechanisms for this thermostability would provide insights into proteins generally and permit the design of synthetic hyperstable biocatalysts.201020487512
essential biological processes of an emerging pathogen: dna replication, transcription, and cell division in acinetobacter spp.within the last 15 years, members of the bacterial genus acinetobacter have risen from relative obscurity to be among the most important sources of hospital-acquired infections. the driving force for this has been the remarkable ability of these organisms to acquire antibiotic resistance determinants, with some strains now showing resistance to every antibiotic in clinical use. there is an urgent need for new antibacterial compounds to combat the threat imposed by acinetobacter spp. and other in ...201020508250
central role of the rna polymerase trigger loop in intrinsic rna hydrolysis.the active center of rna polymerase can hydrolyze phosphodiester bonds in nascent rna, a reaction thought to be important for proofreading of transcription. the reaction proceeds via a general two mg(2+) mechanism and is assisted by the 3' end nucleotide of the transcript. here, by using thermus aquaticus rna polymerase, we show that the reaction also requires the flexible domain of the active center, the trigger loop (tl). we show that the invariant histidine (beta' his1242) of the tl is essent ...201020534498
thermodynamics of the dna structural selectivity of the pol i dna polymerases from escherichia coli and thermus aquaticus.understanding the thermodynamics of substrate selection by dna polymerase i is important for characterizing the balance between replication and repair for this enzyme in vivo. due to their sequence and structural similarities, klenow and klentaq, the large fragments of the pol i dna polymerases from escherichia coli and thermus aquaticus, are considered functional homologs. klentaq, however, does not have a functional proofreading site. examination of the dna binding thermodynamics of klenow and ...201020550914
dmso and betaine greatly improve amplification of gc-rich constructs in de novo synthesis.in synthetic biology, de novo synthesis of gc-rich constructs poses a major challenge because of secondary structure formation and mispriming. while there are many web-based tools for codon optimizing difficult regions, no method currently exists that allows for potentially phenotypically important sequence conservation. therefore, to overcome these limitations in researching gc-rich genes and their non-coding elements, we explored the use of dmso and betaine in two conventional methods of assem ...201020552011
multi-site-specific 16s rrna methyltransferase rsmf from thermus thermophilus.cells devote a significant effort toward the production of multiple modified nucleotides in rrnas, which fine tune the ribosome function. here, we report that two methyltransferases, rsmb and rsmf, are responsible for all four 5-methylcytidine (m(5)c) modifications in 16s rrna of thermus thermophilus. like escherichia coli rsmb, t. thermophilus rsmb produces m(5)c967. in contrast to e. coli rsmf, which introduces a single m(5)c1407 modification, t. thermophilus rsmf modifies three positions, gen ...201020558545
structural basis for the suppression of skin cancers by dna polymerase eta.dna polymerase eta (poleta) is unique among eukaryotic polymerases in its proficient ability for error-free replication through ultraviolet-induced cyclobutane pyrimidine dimers, and inactivation of poleta (also known as polh) in humans causes the variant form of xeroderma pigmentosum (xpv). we present the crystal structures of saccharomyces cerevisiae poleta (also known as rad30) in ternary complex with a cis-syn thymine-thymine (t-t) dimer and with undamaged dna. the structures reveal that the ...201020577207
protein-protein interactions between sigma(70) region 4 of rna polymerase and escherichia coli soxs, a transcription activator that functions by the prerecruitment mechanism: evidence for "off-dna" and "on-dna" interactions.according to the prerecruitment hypothesis, escherichia coli soxs activates the transcription of the genes of the soxrs regulon by forming binary complexes with rna polymerase (rnap) that scan the chromosome for class i and class ii soxs-dependent promoters. we showed previously that the alpha subunit's c-terminal domain plays a role in activating both classes of promoter by making protein-protein contacts with soxs; some of these contacts are made in solution in the absence of promoter dna, a c ...201020595001
promoter melting triggered by bacterial rna polymerase occurs in three steps.rna synthesis, carried out by dna-dependent rna polymerase (rnap) in a process called transcription, involves several stages. in bacteria, transcription initiation starts with promoter recognition and binding of rnap holoenzyme, resulting in the formation of the closed (r.p(c)) rnap-promoter dna complex. subsequently, a transition to the open r.p(o) complex occurs, characterized by separation of the promoter dna strands in an approximately 12 base-pair region to form the transcription bubble. us ...201020615963
a novel heme a insertion factor gene cotranscribes with the thermus thermophilus cytochrome ba3 oxidase locus.studying the biogenesis of the thermus thermophilus cytochrome ba(3) oxidase, we analyze heme a cofactor insertion into this membrane protein complex. only three proteins linked to oxidase maturation have been described for this extreme thermophile, and in particular, no evidence for a canonical surf1 homologue, required for heme a insertion, is available from genome sequence data. here, we characterize the product of an open reading frame, cbax, in the operon encoding subunits of the ba(3)-type ...201020622059
part i: characterization of the extracellular proteome of the extreme thermophile caldicellulosiruptor saccharolyticus by gelc-ms2.the proteome of extremely thermophilic microorganisms affords a glimpse into the dynamics of microbial ecology of high temperature environments. the secretome, or extracellular proteome of these microorganisms, no doubt harbors technologically important enzymes and other thermostable biomolecules that, to date, have been characterized only to a limited extent. in the first of a two-part study on selected thermophiles, defining the secretome requires a sample preparation method that has no negati ...201020623222
active site mutations in mammalian dna polymerase delta alter accuracy and replication fork progression.dna polymerase δ (pol δ) is one of the two main replicative polymerases in eukaryotes; it synthesizes the lagging dna strand and also functions in dna repair. in previous work, we demonstrated that heterozygous expression of the pol δ l604g variant in mice results in normal life span and no apparent phenotype, whereas a different substitution at the same position, l604k, is associated with shortened life span and accelerated carcinogenesis. here, we report in vitro analysis of the homologous mut ...201020628184
functional analysis of the cucumisin propeptide as a potent inhibitor of its mature enzyme.cucumisin is a subtilisin-like serine protease (subtilase) that is found in the juice of melon fruits (cucumis melo l.). it is synthesized as a preproprotein consisting of a signal peptide, nh(2)-terminal propeptide, and 67-kda protease domain. we investigated the role of this propeptide (88 residues) in the cucumisin precursor. complementary dnas encoding the propeptides of cucumisin, two other plant subtilases (arabidopsis ara12 and rice rsp1), and bacterial subtilisin e were expressed in esch ...201020639575
conformational dynamics of bacteriophage t7 dna polymerase and its processivity factor, escherichia coli thioredoxin.gene 5 of bacteriophage t7 encodes a dna polymerase (gp5) responsible for the replication of the phage dna. gp5 polymerizes nucleotides with low processivity, dissociating after the incorporation of 1 to 50 nucleotides. thioredoxin (trx) of escherichia coli binds tightly (kd = 5 nm) to a unique segment in the thumb subdomain of gp5 and increases processivity. we have probed the molecular basis for the increase in processivity. a single-molecule experiment reveals differences in rates of enzymati ...201020696935
a nuclear family a dna polymerase from entamoeba histolytica bypasses thymine glycol.eukaryotic family a dna polymerases are involved in mitochondrial dna replication or translesion dna synthesis. here, we present evidence that the sole family a dna polymerase from the parasite protozoan e. histolytica (ehdnapola) localizes to the nucleus and that its biochemical properties indicate that this dna polymerase may be involved in translesion dna synthesis.201020706627
dna mismatch repair in eukaryotes and bacteria.dna mismatch repair (mmr) corrects mismatched base pairs mainly caused by dna replication errors. the fundamental mechanisms and proteins involved in the early reactions of mmr are highly conserved in almost all organisms ranging from bacteria to human. the significance of this repair system is also indicated by the fact that defects in mmr cause human hereditary nonpolyposis colon cancers as well as sporadic tumors. to date, 2 types of mmrs are known: the human type and escherichia coli type. t ...201020725617
a mutation within the β subunit of escherichia coli rna polymerase impairs transcription from bacteriophage t4 middle promoters.during infection of escherichia coli, bacteriophage t4 usurps the host transcriptional machinery, redirecting it to the expression of early, middle, and late phage genes. middle genes, whose expression begins about 1 min postinfection, are transcribed both from the extension of early rna into middle genes and by the activation of t4 middle promoters. middle-promoter activation requires the t4 transcriptional activator mota and coactivator asia, which are known to interact with σ(70), the specifi ...201020729353
binding of the dimeric deinococcus radiodurans single-stranded dna binding protein to single-stranded dna.deinococcus radiodurans single-stranded (ss) dna binding protein (drssb) originates from a radiation-resistant bacterium and participates in dna recombination, replication, and repair. although it functions as a homodimer, it contains four dna binding domains (ob-folds) and thus is structurally similar to the escherichia coli ssb (ecossb) homotetramer. we examined the equilibrium binding of drssb to ssdna for comparison with that of ecossb. we find that the occluded site size of drssb on poly(dt ...201020795631
unique dna repair gene variations and potential associations with the primary antibody deficiency syndromes igad and cvid.despite considerable effort, the genetic factors responsible for >90% of the antibody deficiency syndromes igad and cvid remain elusive. to produce a functionally diverse antibody repertoire b lymphocytes undergo class switch recombination. this process is initiated by aid-catalyzed deamination of cytidine to uridine in switch region dna. subsequently, these residues are recognized by the uracil excision enzyme ung2 or the mismatch repair proteins mutsalpha (msh2/msh6) and mutlalpha (pms2/mlh1). ...201020805886
twenty-eight divergent polysaccharide loci specifying within- and amongst-strain capsule diversity in three strains of bacteroides fragilis.comparison of the complete genome sequence of bacteroides fragilis 638r, originally isolated in the usa, was made with two previously sequenced strains isolated in the uk (nctc 9343) and japan (ych46). the presence of 10 loci containing genes associated with polysaccharide (ps) biosynthesis, each including a putative wzx flippase and wzy polymerase, was confirmed in all three strains, despite a lack of cross-reactivity between nctc 9343 and 638r surface ps-specific antibodies by immunolabelling ...201020829291
dna polymerase: structural homology, conformational dynamics, and the effects of carcinogenic dna adducts.dna replication is vital for an organism to proliferate and lying at the heart of this process is the enzyme dna polymerase. most dna polymerases have a similar three dimensional fold, akin to a human right hand, despite differences in sequence homology. this structural homology would predict a relatively unvarying mechanism for dna synthesis yet various polymerases exhibit markedly different properties on similar substrates, indicative of each type of polymerase being prescribed to a specific r ...201020847947
identification of critical residues for the tight binding of both correct and incorrect nucleotides to human dna polymerase λ.dna polymerase λ (pol λ) is a novel x-family dna polymerase that shares 34% sequence identity with dna polymerase β. pre-steady-state kinetic studies have shown that the pol λ-dna complex binds both correct and incorrect nucleotides 130-fold tighter, on average, than the dna polymerase β-dna complex, although the base substitution fidelity of both polymerases is 10(-)(4) to 10(-5). to better understand pol λ's tight nucleotide binding affinity, we created single-substitution and double-substitut ...201020851705
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