Publications by authors named "Isaac Wong"

18 Publications

  • Page 1 of 1

Alternative genomic diagnoses for individuals with a clinical diagnosis of Dubowitz syndrome.

Am J Med Genet A 2021 01 24;185(1):119-133. Epub 2020 Oct 24.

Department of Medical Genetics and Alberta Children's Hospital Research Institute, University of Calgary, Calgary, Alberta, Canada.

Dubowitz syndrome (DubS) is considered a recognizable syndrome characterized by a distinctive facial appearance and deficits in growth and development. There have been over 200 individuals reported with Dubowitz or a "Dubowitz-like" condition, although no single gene has been implicated as responsible for its cause. We have performed exome (ES) or genome sequencing (GS) for 31 individuals clinically diagnosed with DubS. After genome-wide sequencing, rare variant filtering and computational and Mendelian genomic analyses, a presumptive molecular diagnosis was made in 13/27 (48%) families. The molecular diagnoses included biallelic variants in SKIV2L, SLC35C1, BRCA1, NSUN2; de novo variants in ARID1B, ARID1A, CREBBP, POGZ, TAF1, HDAC8, and copy-number variation at1p36.11(ARID1A), 8q22.2(VPS13B), Xp22, and Xq13(HDAC8). Variants of unknown significance in known disease genes, and also in genes of uncertain significance, were observed in 7/27 (26%) additional families. Only one gene, HDAC8, could explain the phenotype in more than one family (N = 2). All but two of the genomic diagnoses were for genes discovered, or for conditions recognized, since the introduction of next-generation sequencing. Overall, the DubS-like clinical phenotype is associated with extensive locus heterogeneity and the molecular diagnoses made are for emerging clinical conditions sharing characteristic features that overlap the DubS phenotype.
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http://dx.doi.org/10.1002/ajmg.a.61926DOI Listing
January 2021

Whole exome sequencing analyses reveal gene-microbiota interactions in the context of IBD.

Gut 2021 Feb 10;70(2):285-296. Epub 2020 Jul 10.

Department of Gastroenterology and Hepatology, University of Groningen and University Medical Center Groningen, Groningen, The Netherlands

Objective: Both the gut microbiome and host genetics are known to play significant roles in the pathogenesis of IBD. However, the interaction between these two factors and its implications in the aetiology of IBD remain underexplored. Here, we report on the influence of host genetics on the gut microbiome in IBD.

Design: To evaluate the impact of host genetics on the gut microbiota of patients with IBD, we combined whole exome sequencing of the host genome and whole genome shotgun sequencing of 1464 faecal samples from 525 patients with IBD and 939 population-based controls. We followed a four-step analysis: (1) exome-wide microbial quantitative trait loci (mbQTL) analyses, (2) a targeted approach focusing on IBD-associated genomic regions and protein truncating variants (PTVs, minor allele frequency (MAF) >5%), (3) gene-based burden tests on PTVs with MAF <5% and exome copy number variations (CNVs) with site frequency <1%, (4) joint analysis of both cohorts to identify the interactions between disease and host genetics.

Results: We identified 12 mbQTLs, including variants in the IBD-associated genes , , and . For example, the decrease of the pathway acetyl-coenzyme A biosynthesis, which is involved in short chain fatty acids production, was associated with variants in the gene (false discovery rate <0.05). Changes in functional pathways involved in the metabolic potential were also observed in participants carrying rare PTVs or CNVs in , and genes. These genes are known for their function in the immune system. Moreover, interaction analyses confirmed previously known IBD disease-specific mbQTLs in .

Conclusion: This study highlights that both common and rare genetic variants affecting the immune system are key factors in shaping the gut microbiota in the context of IBD and pinpoints towards potential mechanisms for disease treatment.
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http://dx.doi.org/10.1136/gutjnl-2019-319706DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7815889PMC
February 2021

Dynamic Evolution of Euchromatic Satellites on the X Chromosome in Drosophila melanogaster and the simulans Clade.

Mol Biol Evol 2020 08;37(8):2241-2256

Department of Biology, University of Rochester, Rochester, NY.

Satellite DNAs (satDNAs) are among the most dynamically evolving components of eukaryotic genomes and play important roles in genome regulation, genome evolution, and speciation. Despite their abundance and functional impact, we know little about the evolutionary dynamics and molecular mechanisms that shape satDNA distributions in genomes. Here, we use high-quality genome assemblies to study the evolutionary dynamics of two complex satDNAs, Rsp-like and 1.688 g/cm3, in Drosophila melanogaster and its three nearest relatives in the simulans clade. We show that large blocks of these repeats are highly dynamic in the heterochromatin, where their genomic location varies across species. We discovered that small blocks of satDNA that are abundant in X chromosome euchromatin are similarly dynamic, with repeats changing in abundance, location, and composition among species. We detail the proliferation of a rare satellite (Rsp-like) across the X chromosome in D. simulans and D. mauritiana. Rsp-like spread by inserting into existing clusters of the older, more abundant 1.688 satellite, in events likely facilitated by microhomology-mediated repair pathways. We show that Rsp-like is abundant on extrachromosomal circular DNA in D. simulans, which may have contributed to its dynamic evolution. Intralocus satDNA expansions via unequal exchange and the movement of higher order repeats also contribute to the fluidity of the repeat landscape. We find evidence that euchromatic satDNA repeats experience cycles of proliferation and diversification somewhat analogous to bursts of transposable element proliferation. Our study lays a foundation for mechanistic studies of satDNA proliferation and the functional and evolutionary consequences of satDNA movement.
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http://dx.doi.org/10.1093/molbev/msaa078DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7403614PMC
August 2020

Firefly genomes illuminate parallel origins of bioluminescence in beetles.

Elife 2018 10 16;7. Epub 2018 Oct 16.

Whitehead Institute for Biomedical Research, Cambridge, United States.

Fireflies and their luminous courtships have inspired centuries of scientific study. Today firefly luciferase is widely used in biotechnology, but the evolutionary origin of bioluminescence within beetles remains unclear. To shed light on this long-standing question, we sequenced the genomes of two firefly species that diverged over 100 million-years-ago: the North American and Japanese To compare bioluminescent origins, we also sequenced the genome of a related click beetle, the Caribbean , with bioluminescent biochemistry near-identical to fireflies, but anatomically unique light organs, suggesting the intriguing hypothesis of parallel gains of bioluminescence. Our analyses support independent gains of bioluminescence in fireflies and click beetles, and provide new insights into the genes, chemical defenses, and symbionts that evolved alongside their luminous lifestyle.
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http://dx.doi.org/10.7554/eLife.36495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191289PMC
October 2018

A data-driven approach for addressing the lack of flow waveform data in studies of cerebral arterial flow in older adults.

Physiol Meas 2018 02 1;39(1):015006. Epub 2018 Feb 1.

Department of Mechanical Engineering and Materials Science, University of Pittsburgh, Pittsburgh, Pennsylvania, United States of America.

Objective: Blood flow waveforms-essential data for hemodynamic modeling-are often in practice unavailable to researchers. The objectives of this work were to assess the variability among the waveforms for a clinically relevant older population, and develop data-based methods for addressing the missing waveform data for hemodynamic studies.

Approach: We analyzed 272 flow waveforms from the internal carotid arteries of older patients (73  ±  13 yr) with moderate cardiovascular disease, and used these data to develop methods to guide new approaches for hemodynamic studies.

Main Results: Profound variations in waveform parameters were found within the aged population that were not seen in published data for young subjects. Common features in the aged population relative to the young included a larger systole-to-diastole flow rate ratio, increased flow during late systole, and absence of a dicrotic notch. Eight waveforms were identified that collectively represent the range of waveforms in the older population. A relationship between waveform shape and flow rate was obtained that, in conjunction with equations relating flow rate to diameter, can be used to provide individualized waveforms for patient-specific geometries. The dependence of flow rate on diameter was statistically different between male and female patients.

Significance: It was shown that a single archetypal waveform cannot well-represent the diverse waveforms found within an aged population, although this approach is frequently used in studies of flow in the cerebral vasculature. Motivated by these results, we provided a set of eight waveforms that can be used to assess the hemodynamic uncertainty associated with the lack of patient-specific waveform data. We also provided a methodology for generating individualized waveforms when patient gender, age, and cardiovascular disease state are known. These data-driven approaches can be used to devise more relevant in vitro or in silico intra-cranial hemodynamic studies for older patients.
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http://dx.doi.org/10.1088/1361-6579/aa9f46DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5811231PMC
February 2018

A Rapid and Low-Cost PCR Thermal Cycler for Low Resource Settings.

PLoS One 2015 6;10(7):e0131701. Epub 2015 Jul 6.

AI Biosciences, Inc., College Station, Texas, United States of America.

Background: Many modern molecular diagnostic assays targeting nucleic acids are typically confined to developed countries or to the national reference laboratories of developing-world countries. The ability to make technologies for the rapid diagnosis of infectious diseases broadly available in a portable, low-cost format would mark a revolutionary step forward in global health. Many molecular assays are also developed based on polymerase chain reactions (PCR), which require thermal cyclers that are relatively heavy (>20 pounds) and need continuous electrical power. The temperature ramping speed of most economical thermal cyclers are relatively slow (2 to 3 °C/s) so a polymerase chain reaction can take 1 to 2 hours. Most of all, these thermal cyclers are still too expensive ($2k to $4k) for low-resource setting uses.

Methodology/principal Findings: In this article, we demonstrate the development of a low-cost and rapid water bath based thermal cycler that does not require active temperature control or continuous power supply during PCR. This unit costs $130 to build using commercial off-the-shelf items. The use of two or three vacuum-insulated stainless-steel Thermos food jars containing heated water (for denaturation and annealing/extension steps) and a layer of oil on top of the water allow for significantly stabilized temperatures for PCR to take place. Using an Arduino-based microcontroller, we automate the "archaic" method of hand-transferring PCR tubes between water baths.

Conclusions/significance: We demonstrate that this innovative unit can deliver high speed PCR (17 s per PCR cycle) with the potential to go beyond the 1,522 bp long amplicons tested in this study and can amplify from templates down to at least 20 copies per reaction. The unit also accepts regular PCR tubes and glass capillary tubes. The PCR efficiency of our thermal cycler is not different from other commercial thermal cyclers. When combined with a rapid nucleic acid detection approach, the thermos thermal cycler (TTC) can enable on-site molecular diagnostics in low-resource settings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131701PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4492969PMC
April 2016

Land use change impacts on water quality in three lake winnipeg watersheds.

J Environ Qual 2014 Sep;43(5):1690-701

Lake Winnipeg eutrophication results from excess nutrient loading due to agricultural activities across the watershed. Estimating nonpoint-source pollution and the mitigation effects of beneficial management practices (BMPs) is an important step in protecting the water quality of streams and receiving waters. The use of computer models to systematically compare different landscapes and agricultural systems across the Red-Assiniboine basin has not been attempted at watersheds of this size in Manitoba. In this study, the Soil and Water Assessment Tool was applied and calibrated for three pilot watersheds of the Lake Winnipeg basin. Monthly flow calibration yielded overall satisfactory Nash-Sutcliffe efficiency (NSE), with values above 0.7 for all simulations. Total phosphorus (TP) calibration NSE ranged from 0.64 to 0.76, total N (TN) ranged from 0.22 to 0.75, and total suspended solids (TSS) ranged from 0.29 to 0.68. Based on the assessment of the TP exceedance levels from 1993 to 2007, annual loads were above proposed objectives for the three watersheds more than half of the time. Four BMP scenarios based on land use changes were studied in the watersheds: annual cropland to hay land (ACHL), wetland restoration (WR), marginal annual cropland conversion to hay land (MACHL), and wetland restoration on marginal cropland (WRMAC). Of these land use change scenarios, ACHL had the greatest impact: TSS loads were reduced by 33 to 65%, TN by 58 to 82%, and TP by 38 to 72% over the simulation period. By analyzing unit area and percentage of load reduction, the results indicate that the WR and WRMAC scenarios had a significant impact on water quality in high loading zones in the three watersheds. Such reductions of sediment, N, and P are possible through land use change scenarios, suggesting that land conservation should be a key component of any Lake Winnipeg restoration strategy.
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http://dx.doi.org/10.2134/jeq2013.06.0234DOI Listing
September 2014

Loop202-208 in avian sarcoma virus integrase mediates tetramer assembly and processing activity.

Biochemistry 2007 Oct 11;46(40):11231-9. Epub 2007 Sep 11.

Department of Molecular and Cellular Biochemistry, University of Kentucky, Lexington, Kentucky 40536, USA.

Integrase (IN) catalyzes insertion of the retroviral genome into the host via two sequential reactions. The processing activity cleaves the 3'-dinucleotides from the two ends of the viral DNA which are then inserted into the host DNA. Tetramers are required for the joining step. While dimers have been shown to catalyze processing, they do so inefficiently, and the oligomeric requirement for processing is unknown. We have replaced loop202-208 at the putative dimer-dimer interface of the avian sarcoma virus IN with its analogue, loop188-194, from human immunodeficiency virus IN. The mutation abolished disintegration activity and a 2 x 10(-2) s-1 fast phase during single-turnover processing. A 3 x 10(-4) s-1 slow processing phase was unaffected. Preincubation with a DNA substrate known to promote tetramerization increased products formed during the fast phase by 2.5-fold only for wild-type IN, correlating the fast and slow phases with processing by tetramers and dimers, respectively. We propose a novel tetramer model for coupling processing and integration based on efficient processing by the tetramer. We provide for the first time direct evidence of the functional relevance of a structural element, loop202-208, which appears to be required for mediating the interaction between dimer halves of the active tetramer.
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http://dx.doi.org/10.1021/bi700197aDOI Listing
October 2007

Investigating the geminal diamine intermediate of Yersinia pestis arginine decarboxylase with substrate, product, and inhibitors using single wavelength stopped-flow spectroscopy.

Biochemistry 2007 Jan;46(2):379-86

Department of Pharmaceutical Sciences, College of Pharmacy, University of Kentucky, Lexington, USA.

The reaction mechanism of Yersinia pestis arginine decarboxylase has been investigated using a series of substrate, product, and inhibitors. Using single wavelength stopped-flow spectroscopy, novel mechanistic features were noted in the presence of the product, agmatine. By focusing on the excitation and emission wavelengths of the geminal diamine intermediate, we were able to monitor the formation and decay of two different geminal diamine species. Experiments revealed that the enzyme exists in two different conformational states--one that binds ligand and one that does not. The on and off rates for the conversion between the two conformational states was determined to be 390 s-1 and 880 s-1, respectively. The KD for agmatine binding was 6 mM. In addition, experiments revealed a pH-dependent conversion between two states of the enzyme. The deprotonated form of the enzyme binds ligand more slowly than the protonated form. The rates for the formation of the geminal diamine and external aldimine in this pathway were determined to be 25 and 4 s-1, respectively. There is also a slow interconversion between the protonated and deprotonated enzymes that has a pKa of approximately 8.0. Finally, the formation of the geminal diamine was determined to be Mg2+-dependent.
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http://dx.doi.org/10.1021/bi061260hDOI Listing
January 2007

Regulation of replication protein A functions in DNA mismatch repair by phosphorylation.

J Biol Chem 2006 Aug 26;281(31):21607-21616. Epub 2006 May 26.

Department of Molecular & Cellular Biochemistry and Markey Cancer Center, University of Kentucky Medical Center, Lexington, Kentucky 40536; Graduate Center for Toxicology and Department of Pathology, University of Kentucky Medical Center, Lexington, Kentucky 40536. Electronic address:

Replication protein A (RPA) is involved in multiple stages of DNA mismatch repair (MMR); however, the modulation of its functions between different stages is unknown. We show here that phosphorylation likely modulates RPA functions during MMR. Unphosphorylated RPA initially binds to nicked heteroduplex DNA to facilitate assembly of the MMR initiation complex. The unphosphorylated protein preferentially stimulates mismatch-provoked excision, possibly by cooperatively binding to the resultant single-stranded DNA gap. The DNA-bound RPA begins to be phosphorylated after extensive excision, resulting in severalfold reduction in the DNA binding affinity of RPA. Thus, during the phase of repair DNA synthesis, the phosphorylated RPA readily disassociates from DNA, making the DNA template available for DNA polymerase delta-catalyzed resynthesis. These observations support a model of how phosphorylation alters the DNA binding affinity of RPA to fulfill its differential requirement at the various stages of MMR.
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http://dx.doi.org/10.1074/jbc.M603504200DOI Listing
August 2006

Development of a new approach to cumulative effects assessment: a northern river ecosystem example.

Environ Monit Assess 2006 Feb;113(1-3):87-115

National Water Research Institute (NWRI), Environment Canada, Saskatoon, SK.

If sustainable development of Canadian waters is to be achieved, a realistic and manageable framework is required for assessing cumulative effects. The objective of this paper is to describe an approach for aquatic cumulative effects assessment that was developed under the Northern Rivers Ecosystem Initiative. The approach is based on a review of existing monitoring practices in Canada and the presence of existing thresholds for aquatic ecosystem health assessments. It suggests that a sustainable framework is possible for cumulative effects assessment of Canadian waters that would result in integration of national indicators of aquatic health, integration of national initiatives (e.g., water quality index, environmental effects monitoring), and provide an avenue where long-term monitoring programs could be integrated with baseline and follow-up monitoring conducted under the environmental assessment process.
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http://dx.doi.org/10.1007/s10661-005-9098-0DOI Listing
February 2006

Assessing potential for recovery of biotic richness and indicator species due to changes in acidic deposition and lake pH in five areas of southeastern Canada.

Environ Monit Assess 2003 Oct-Nov;88(1-3):53-101

Fisheries and Oceans Canada, Great Lakes Laboratory for Fisheries & Aquatic Sciences, 867 Lakeshore Rd., Box 5050, Burlington, ON, Canada, L7R 4A6.

Biological damage to sensitive aquatic ecosystems is among the most recognisable, deleterious effects of acidic deposition. We compiled a large spatial database of over 2000 waterbodies across southeastern Canada from various federal, provincial and academic sources. Data for zooplankton, fish, macroinvertebrate (benthos) and loon species richness and occurrence were used to construct statistical models for lakes with varying pH, dissolved organic carbon content and lake size. pH changes, as described and predicted using the Integrated Assessment Model (Lam et al., 1998; Jeffries et al., 2000), were based on the range of emission reductions set forth in the Canada/US Air Quality Agreement (AQA). The scenarios tested include 1983, 1990, 1994 and 2010 sulphate deposition levels. Biotic models were developed for five regions in southeastern Canada (Algoma, Muskoka, and Sudbury, Ontario, southcentral Quebec, and Kejimkujik, Nova Scotia) using regression tree, multiple linear regression and logistic regression analyses to make predictions about recovery after emission reductions. The analyses produced different indicator species in different regions, although some species showed consistent trends across regions. Generally, the greatest predicted recovery occurred during the final phase of emission reductions between 1994 and 2010 across all taxonomic groups and regions. The Ontario regions, on average, were predicted to recover to a greater extent than either southcentral Quebec or the Kejimkujik area of Nova Scotia. Our results reconfirm that pH 5.5-6.0 is an important threshold below which damage to aquatic biota will remain a major local and regional environmental problem. This damage to biodiversity across trophic levels will persist well into the future if no further reductions in sulphate deposition are implemented.
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http://dx.doi.org/10.1023/a:1025548518991DOI Listing
February 2004

Functional oligomeric state of avian sarcoma virus integrase.

J Biol Chem 2003 Jan 21;278(2):1323-7. Epub 2002 Nov 21.

Department of Biochemistry and Biophysics, Oregon State University, Oregon 97331, USA.

Retroviral integrase, one of only three enzymes encoded by the virus, catalyzes the essential step of inserting a DNA copy of the viral genome into the host during infection. Using the avian sarcoma virus integrase, we demonstrate that the enzyme functions as a tetramer. In presteady-state active site titrations, four integrase protomers were required for a single catalytic turnover. Volumetric determination of integrase-DNA complexes imaged by atomic force microscopy during the initial turnover additionally revealed substrate-induced assembly of a tetramer. These results suggest that tetramer formation may be a requisite step during catalysis with ramifications for antiviral design strategies targeting the structurally homologous human immunodeficiency virus, type 1 (HIV-1) integrase.
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http://dx.doi.org/10.1074/jbc.C200550200DOI Listing
January 2003

A dimeric mechanism for contextual target recognition by MutY glycosylase.

J Biol Chem 2003 Jan 18;278(4):2411-8. Epub 2002 Nov 18.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis 97331, USA.

MutY, an adenine glycosylase, initiates the critical repair of an adenine:8-oxo-guanine base pair in DNA arising from polymerase error at the oxidatively damaged guanine. Here we demonstrate for the first time, using presteady-state active site titrations, that MutY assembles into a dimer upon binding substrate DNA and that the dimer is the functionally active form of the enzyme. Additionally, we observed allosteric inhibition of glycosylase activity in the dimer by the concurrent binding of two lesion mispairs. Active site titration results were independently verified by gel mobility shift assays and quantitative DNA footprint titrations. A model is proposed for the potential functional role of the observed polysteric and allosteric regulation in recruiting and coordinating interactions with the methyl-directed mismatch repair system.
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http://dx.doi.org/10.1074/jbc.M209802200DOI Listing
January 2003

Flipping duplex DNA inside out: a double base-flipping reaction mechanism by Escherichia coli MutY adenine glycosylase.

J Biol Chem 2002 Jun 18;277(23):20960-4. Epub 2002 Apr 18.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.

The Escherichia coli MutY adenine glycosylase plays a critical role in repairing mismatches in DNA between adenine and the oxidatively damaged guanine base 8-oxoguanine. Crystallographic studies of the catalytic core domain of MutY show that the scissile adenine is extruded from the DNA helix to be bound in the active site of the enzyme (Guan, Y., Manuel, R. C., Arvai, A. S., Parikh, S. S., Mol, C. D., Miller, J. H., Lloyd, S., and Tainer, J. A. (1998) Nat. Struct. Biol. 5, 1058-1064). However, the structural and mechanistic bases for the recognition of the 8-oxoguanine remain poorly understood. In experiments using a single-stranded 8-bromoguanine-containing synthetic oligodeoxyribonucleotide alone and in a duplex construct mismatched to an adenine, we observed UV cross-linking between MutY and the 8-bromoguanine probe. We further observed enhanced cross-linking in the single strand experiments, suggesting that neither the duplex context nor the mismatch with adenine is required for recognition of the 8-oxoguanine moiety. Stopped-flow fluorescence studies using 2-aminopurine-containing oligodeoxyribonucleotides further revealed the sequential extrusion of the 8-oxoguanine at 108 s(-1) followed by the adenine at 16 s(-1). A protein isomerization step following base flipping at 1.9 s(-1) was also observed and is postulated to provide additional stabilization of the extruded adenine thereby facilitating its capture by the active site for excision.
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http://dx.doi.org/10.1074/jbc.C200181200DOI Listing
June 2002

Presteady-state analysis of a single catalytic turnover by Escherichia coli uracil-DNA glycosylase reveals a "pinch-pull-push" mechanism.

J Biol Chem 2002 May 20;277(22):19424-32. Epub 2002 Mar 20.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.

Uracil-DNA glycosylase catalyzes the excision of uracils from DNA via a mechanism where the uracil is extrahelically flipped out of the DNA helix into the enzyme active site. A conserved leucine is inserted into the DNA duplex space vacated by the uracil leading to the paradigmatic "push-pull" mechanism of nucleotide flipping. However, the order of these two steps during catalysis has not been conclusively established. We report a complete kinetic analysis of a single catalytic turnover using a hydrolyzable duplex oligodeoxyribonucleotide substrate containing a uracil:2-aminopurine base pair. Rapid chemical-quenched-flow methods defined the kinetics of excision at the active site during catalysis. Stopped-flow fluorometry monitoring the 2-aminopurine fluorescence defined the kinetics of uracil flipping. Parallel experiments detecting the protein fluorescence showed a slower Leu(191) insertion step occurring after nucleotide flipping but before excision. The inserted Leu(191) acts as a doorstop to prevent the return of the flipped-out uracil residue, thereby facilitating the capture of the uracil in the active site and does not play a direct role in "pushing" the uracil out of the DNA helix. The results define for the first time the proper sequence of events during a catalytic cycle and establish a "pull-push", as opposed to a "push-pull", mechanism for nucleotide flipping.
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http://dx.doi.org/10.1074/jbc.M201198200DOI Listing
May 2002

Presteady-state analysis of avian sarcoma virus integrase. I. A splicing activity and structure-function implications for cognate site recognition.

J Biol Chem 2002 Apr 30;277(14):12089-98. Epub 2002 Jan 30.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.

Integrase catalyzes insertion of a retroviral genome into the host chromosome. After reverse transcription, integrase binds specifically to the ends of the duplex retroviral DNA, endonucleolytically cleaves two nucleotides from each 3'-end (the processing activity), and inserts these ends into the host DNA (the joining activity) in a concerted manner. In first-turnover experiments with synapsed DNA substrates, we observed a novel splicing activity that resembles an integrase joining reaction but uses unprocessed ends. This splicing reaction showed an initial exponential phase (k(splicing) = 0.02 s(-1)) of product formation and generated products macroscopically indistinguishable from those created by the processing and joining activities, thus bringing into question methods previously used to quantitate these reactions in a time regime where multiple turnovers of the enzyme have occurred. With a presteady-state assay, however, we were able to distinguish between different pathways that led to formation of identical products. Furthermore, the splicing reaction allowed characterization of substrate binding and specificity. Although integrase requires only a 3' hydroxyl with respect to nucleophiles derived from DNA, it specifically favors the cognate sequence CATT as the electrophile. These experimental results support a two-site "switching" model for binding and catalysis of all three integrase activities.
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http://dx.doi.org/10.1074/jbc.M111315200DOI Listing
April 2002

Presteady-state analysis of avian sarcoma virus integrase. II. Reverse-polarity substrates identify preferential processing of the U3-U5 pair.

J Biol Chem 2002 Apr 30;277(14):12099-108. Epub 2002 Jan 30.

Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, USA.

The integrase-catalyzed insertion of the retroviral genome into the host chromosome involves two reactions in vivo: 1) the binding and endonucleolytic removal of the terminal dinucleotides of the viral DNA termini and 2) the recombination of the ends with the host DNA. Kukolj and Skalka (Kukolj, G., and Skalka, A. M. (1995) Genes Dev. 9, 2556-2567) have previously shown that tethering of the termini enhances the endonucleolytic activities of integrase. We have used 5'-5' phosphoramidites to design reverse-polarity tethers that allowed us to examine the reactivity of two viral long terminal repeat-derived sequences when concurrently bound to integrase and, additionally, developed presteady-state assays to analyze the initial exponential phase of the reaction, which is a measure of the amount of productive nucleoprotein complexes formed during preincubation of integrase and DNA. Furthermore, the reverse-polarity tether circumvents the integrase-catalyzed splicing reaction (Bao, K., Skalka, A. M., and Wong, I. (2002) J. Biol. Chem. 277, 12089-12098) that obscures accurate analysis of the reactivities of synapsed DNA substrates. Consequently, we were able to establish a lower limit of 0.2 s(-1) for the rate constant of the processing reaction. The analysis showed the physiologically relevant U3/U5 pair of viral ends to be the preferred substrate for integrase with the U3/U3 combination favored over the U5/U5 pair.
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http://dx.doi.org/10.1074/jbc.M111314200DOI Listing
April 2002