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1.  Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase 
Nucleic Acids Research  2013;42(3):1831-1844.
Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10−3 s−1 and KM < 1 nM at 25°C under conditions where T4 DNA ligase produced only 5′-adenylylated DNA with a 20-fold lower kcat and a KM ≈ 300 nM. The rate of ligation increased with addition of Mn2+, but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5′-phosphorylated dC or dG residue on the 3′ side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
PMCID: PMC3919565  PMID: 24203707
2.  A network of PUF proteins and Ras signaling promote mRNA repression and oogenesis in C. elegans 
Developmental Biology  2012;366(2):218-231.
Cell differentiation requires integration of gene expression controls with dynamic changes in cell morphology, function, and control. Post-transcriptional mRNA regulation and signaling systems are important to this process but their mechanisms and connections are unclear. During C. elegans oogenesis, we find that two groups of PUF RNA binding proteins (RNABPs), PUF-3/11 and PUF-5/6/7, control different specific aspects of oocyte formation. PUF-3/11 limits oocyte growth, while PUF-5/6/7 promotes oocyte organization and formation. These two PUF groups repress mRNA translation through overlapping but distinct sets of 3’ untranslated regions (3’UTRs). Several PUF-dependent mRNAs encode other mRNA regulators suggesting both PUF groups control developmental patterning of mRNA regulation circuits. Furthermore, we find that the Ras-MapKinase/ERK pathway functions with PUF-5/6/7 to repress specific mRNAs and control oocyte organization and growth. These results suggest that diversification of PUF proteins and their integration with Ras-MAPK signaling modulates oocyte differentiation. Together with other studies, these findings suggest positive and negative interactions between the Ras-MAPK system and PUF RNA-binding proteins likely occur at multiple levels. Changes in these interactions over time can influence spatiotemporal patterning of tissue development.
PMCID: PMC3361503  PMID: 22542599
Translational control; PUF protein; Oogenesis; RNA-binding protein; Ras; Map Kinase; ERK; translation
3.  A Fast Solution to NGS Library Prep with Low Nanogram DNA Input 
Next Generation Sequencing (NGS) has significantly impacted human genetics, enabling a comprehensive characterization of the human genome as well as a better understanding of many genomic abnormalities. By delivering massive DNA sequences at unprecedented speed and cost, NGS promises to make personalized medicine a reality in the foreseeable future. To date, library construction with clinical samples has been a challenge, primarily due to the limited quantities of sample DNA available. Our objective here was to overcome this challenge by developing NEBNext® Ultra DNA Library Prep Kit, a fast library preparation method. Specifically, we streamlined the workflow utilizing novel NEBNext reagents and adaptors, including a new DNA polymerase that has been optimized to minimize GC bias. As a result of this work, we have developed a simple method for library construction from an amount of DNA as low as 5 ng, which can be used for both intact and fragmented DNA. Moreover, the workflow is compatible with multiple NGS platforms.
PMCID: PMC3635320
4.  Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification 
In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.
Author Summary
Brugian filariasis is a debilitating neglected tropical disease caused by infection with the filarial parasites Brugia malayi or Brugia timori. Adult worms live in the lymphatic system and produce large numbers of microfilariae that predominantly circulate in the blood at night. Bloodsucking mosquitoes spread the disease by ingesting microfilariae that develop into infective stage larvae in the insect. In rural areas, diagnosis still relies largely on microscopic examination of night blood and morphological assessment of stained microfilariae. Loop-mediated isothermal amplification (LAMP) is a technique that can amplify DNA with high specificity, sensitivity and rapidity under isothermal conditions. The operational simplicity, versatility and low-cost of the technique make it particularly appealing for use in diagnosis and geographical mapping of neglected tropical diseases. In the present study, we have developed and evaluated a Brugia Hha I repeat LAMP assay for the rapid detection of B. malayi and B. timori genomic DNA. The results indicate that the Brugia Hha I repeat LAMP diagnostic assay is sensitive and rapid, detecting a single microfilariae in blood within 30 minutes, and Brugia-specific. The test has the potential to be developed further as a field tool for use in the implementation and management of mass drug administration programs for brugian filariasis.
PMCID: PMC3521703  PMID: 23272258
6.  Maternal mRNAs are regulated by diverse P body–related mRNP granules during early Caenorhabditis elegans development 
The Journal of Cell Biology  2008;182(3):559-572.
Processing bodies (P bodies) are conserved mRNA–protein (mRNP) granules that are thought to be cytoplasmic centers for mRNA repression and degradation. However, their specific functions in vivo remain poorly understood. We find that repressed maternal mRNAs and their regulators localize to P body–like mRNP granules in the Caenorhabditis elegans germ line. Surprisingly, several distinct types of regulated granules form during oocyte and embryo development. 3′ untranslated region elements direct mRNA targeting to one of these granule classes. The P body factor CAR-1/Rap55 promotes association of repressed mRNA with granules and contributes to repression of Notch/glp-1 mRNA. However, CAR-1 controls Notch/glp-1 only during late oogenesis, where it functions with the RNA-binding regulators PUF-5, PUF-6, and PUF-7. The P body protein CGH-1/Rck/Dhh1 differs from CAR-1 in control of granule morphology and promotes mRNP stability in arrested oocytes. Therefore, a system of diverse and regulated RNP granules elicits stage-specific functions that ensure proper mRNA control during early development.
PMCID: PMC2500140  PMID: 18695046
7.  Kinetic Characterization of Single Strand Break Ligation in Duplex DNA by T4 DNA Ligase 
The Journal of Biological Chemistry  2011;286(51):44187-44196.
Background: T4 DNA ligase catalyzes the formation of phosphodiester bonds in dsDNA.
Results: Single turnover rates of all chemical reaction steps exceed steady state turnover rates by 10-fold.
Conclusion: Product release or a postligation conformational change is rate-limiting during turnover.
Significance: This study represents the first detailed analysis of the kinetic mechanism of nick ligation by T4 DNA ligase.
T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5′-phosphate and 3′-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5′-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5′-phosphate or a 5′-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5′-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4 ± 0.1 s−1 and 150 ± 50 μm−1 s−1, respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded kcat and kcat/Km values of 0.64 ± 0.08 s−1 and 240 ± 40 μm−1 s−1. Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s−1, respectively, with the slowest step ∼10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s−1, also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (∼6 s−1), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion.
PMCID: PMC3243518  PMID: 22027837
DNA Enzymes; DNA Repair; DNA-Protein Interaction; Enzyme Kinetics; Enzyme Mechanisms; Pre-steady State Kinetics; Rapid Quench-Flow; T4 DNA Ligase
8.  A Human PMS2 Homologue from Aquifex aeolicus Stimulates an ATP-dependent DNA Helicase 
The Journal of Biological Chemistry  2010;285(15):11087-11092.
Mismatch repair in Escherichia coli involves a number of proteins including MutL and UvrD. Eukaryotes also possess MutL homologues; however, no UvrD helicase homologues have been identified. The hyperthermophilic bacterium Aquifex aeolicus has a MutL protein (Aae MutL) that possesses a latent endonuclease activity similar to eukaryotic, but different from E. coli, MutL proteins. By sequence homology Aq793 is a member of the PcrA/UvrD/Rep helicase subfamily. We expressed Aae MutL and the putative A. aeolicus DNA helicase (Aq793) proteins in E. coli. Using synthetic oligonucleotide substrates, we observed that lower concentrations of Aq793 were required to unwind double-stranded DNA that had a 3′-poly(dT) overhang as compared with double-stranded DNA with a 5′-poly(dT) or lacking a poly(dT) tail. This unwinding activity was stimulated by adding Aae MutL with maximal stimulation observed at an ∼1.5:1 (MutL:Aq793) stoichiometric ratio. The enhancement of Aq793 helicase activity did not require the Aae MutL protein to retain endonuclease activity. Furthermore, the C-terminal 123 amino acid residues of Aae MutL were sufficient to stimulate Aq793 helicase activity, albeit at a significantly reduced efficacy. To the best of our knowledge this is the first time a human PMS2 homologue has been demonstrated to stimulate a PcrA/UvrD/Rep subfamily helicase, and this finding may further our understanding of the evolution of the mismatch repair pathway.
PMCID: PMC2856984  PMID: 20129926
DNA/Damage; DNA/Enzymes; DNA/Helicase; DNA/Protein Interaction; DNA/Repair; DNA/Uvr ABCD System; MutL; Helicase
9.  Adenosine Triphosphate Stimulates Aquifex aeolicus MutL Endonuclease Activity 
PLoS ONE  2009;4(9):e7175.
Human PMS2 (hPMS2) homologues act to nick 5′ and 3′ to misincorporated nucleotides during mismatch repair in organisms that lack MutH. Mn++ was previously found to stimulate the endonuclease activity of these homologues. ATP was required for the nicking activity of hPMS2 and yPMS1, but was reported to inhibit bacterial MutL proteins from Thermus thermophilus and Aquifex aeolicus that displayed homology to hPMS2. Mutational analysis has identified the DQHA(X)2E(X)4E motif present in the C-terminus of PMS2 homologues as important for endonuclease activity.
Methodologies/Principal Findings
We examined the effect ATP had on the Mn++ induced nicking of supercoiled pBR322 by full-length and mutant A. aeolicus MutL (Aae MutL) proteins. Assays were single time point, enzyme titration experiments or reaction time courses. The maximum velocity for MutL nicking was determined to be 1.6±0.08×10−5 s−1 and 4.2±0.3×10−5 s−1 in the absence and presence of ATP, respectively. AMPPNP stimulated the nicking activity to a similar extent as ATP. A truncated Aae MutL protein composed of only the C-terminal 123 amino acid residues was found to nick supercoiled DNA. Furthermore, mutations in the conserved C-terminal DQHA(X)2E(X)4E and CPHGRP motifs were shown to abolish Aae MutL endonuclease activity.
ATP stimulated the Mn++ induced endonuclease activity of Aae MutL. Experiments utilizing AMPPNP implied that the stimulation did not require ATP hydrolysis. A mutation in the DQHA(X)2E(X)4E motif of Aae MutL further supported the role of this region in endonclease activity. For the first time, to our knowledge, we demonstrate that changing the histidine residue in the conserved CPHGRP motif abolishes endonucleolytic activity of a hPMS2 homologue. Finally, the C-terminal 123 amino acid residues of Aae MutL were sufficient to display Mn++ induced nicking activity.
PMCID: PMC2744016  PMID: 19777055
10.  DNA damage in preserved specimens and tissue samples: a molecular assessment 
Frontiers in Zoology  2008;5:18.
The extraction of genetic information from preserved tissue samples or museum specimens is a fundamental component of many fields of research, including the Barcode of Life initiative, forensic investigations, biological studies using scat sample analysis, and cancer research utilizing formaldehyde-fixed, paraffin-embedded tissue. Efforts to obtain genetic information from these sources are often hampered by an inability to amplify the desired DNA as a consequence of DNA damage.
Previous studies have described techniques for improved DNA extraction from such samples or focused on the effect of damaging agents – such as light, oxygen or formaldehyde – on free nucleotides.
We present ongoing work to characterize lesions in DNA samples extracted from preserved specimens. The extracted DNA is digested to single nucleosides with a combination of DNase I, Snake Venom Phosphodiesterase, and Antarctic Phosphatase and then analyzed by HPLC-ESI-TOF-MS.
We present data for moth specimens that were preserved dried and pinned with no additional preservative and for frog tissue samples that were preserved in either ethanol, or formaldehyde, or fixed in formaldehyde and then preserved in ethanol. These preservation methods represent the most common methods of preserving animal specimens in museum collections. We observe changes in the nucleoside content of these samples over time, especially a loss of deoxyguanosine. We characterize the fragmentation state of the DNA and aim to identify abundant nucleoside lesions. Finally, simple models are introduced to describe the DNA fragmentation based on nicks and double-strand breaks.
PMCID: PMC2579423  PMID: 18947416
11.  Reconstitution of Glyphosate Resistance from a Split 5-Enolpyruvyl Shikimate-3-Phosphate Synthase Gene in Escherichia coli and Transgenic Tobacco▿ †  
Applied and Environmental Microbiology  2007;73(24):7997-8000.
A highly N-phosphonomethylglycine (glyphosate)-resistant Pseudomonas fluorescens G2 5-enolpyruvyl shikimate-3-phosphate synthase (EPSPS) was mapped to identify potential split sites using a transposon-based linker-scanning procedure. Intein-mediated protein complementation was used to reconstitute glyphosate resistance from the genetically divided G2 EPSPS gene in Escherichia coli strain ER2799 and transgenic tobacco.
PMCID: PMC2168149  PMID: 17951442

Results 1-11 (11)