Robert Kuchta - Academia.edu (original) (raw)

Papers by Robert Kuchta

Research paper thumbnail of Fe-S centers in lactyl-CoA dehydratase

Biochemistry, Nov 1, 1986

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of A... more Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Biochemistry Usa, Mar 1, 1991

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleo... more Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of D N A polymerase I [KF(exo+)].

Research paper thumbnail of Interactions of DNA with Human DNA Primase Monitored with Photoactivatable Cross-Linking Agents: Implications for the Role of the p58 Subunit †

Biochemistry Usa, Oct 1, 1999

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the prim... more Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.

Research paper thumbnail of Dna array analysis as a diagnostic for current and emerging strains of influenza

Research paper thumbnail of Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

Biomacromolecules, Mar 1, 2010

This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sens... more This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm 2 . Film heights of 10-20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm 2 while below the detection limit of ~10 EITC-nucleotides/μm 2 , no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges.

Research paper thumbnail of Detection of Antiviral Resistance in Influenza a Using Dna Microarray

Research paper thumbnail of Herpes Simplex Virus-1 Helicase−Primase: Roles of Each Subunit in DNA Binding and Phosphodiester Bond Formation

Biochemistry Usa, Sep 1, 2009

The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and ... more The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two subunit complexes, UL5/UL52, UL5/UL8, and UL52/ UL8, and used a series of kinetic and photocrosslinking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5/UL52 by increasing the rate of primer initiation (2 NTPs → pppNpN), the rate-limiting step in primer synthesis. The UL5/UL8 complex lacked any detectable catalytic activity (DNA dependent ATPase, primase, or RNA polymerase using a RNA primer:template and NTPs as substrates), but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52/UL8 complex lacked detectable DNA dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer:template and NTPs as substrate. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for initiation of primer synthesis. Photocrosslinking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3′-GPyPy (Py = T or C) initiation site for primer synthesis showed that only UL5 crosslinked to the DNA. This occurred for both the UL5/UL52 and UL5/UL52/UL8 complexes, and whether or not the reactions contained NTPs. Photocrosslinking of a RNA primer:template, the product of primer synthesis, containing 5iodouracil in the template generated the same apparent crosslinked species.

Research paper thumbnail of Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza AB Test for Rapid Diagnosis of Influenza

The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blin... more The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A؉B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.

Research paper thumbnail of Mechanism of Primer Synthesis by the Herpes Simplex Virus 1 Helicase-Primase �

Biochemistry Usa, Mar 1, 2004

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by her... more We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.

Research paper thumbnail of The Technology Transfer Awards honor faculty, inventors, business advisors and entrepreneurs who demonstrate best practices in the technology transfer cycle

Research paper thumbnail of Hall, E. T., Yan, J. P., Melancon, P. & Kuchta, R. D. 3'-Azido-3'-deoxythymidine potently inhibits protein glycosylation. A novel mechanism for AZT cytotoxicity. J. Biol. Chem. 269, 14355-1438

Journal of Biological Chemistry

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatm... more 3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (W infection. Unfor-tunateIy, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythpidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This i~i b i t i o n results from direct competition with several pyrimidinesugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve tl-azido-3"deoxythpidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects, thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.

Research paper thumbnail of Interactions of calf thymus DNA polymerase alpha with primer/templates

Nucleic Acids Research

The interactions of calf thymus DNA polymerase a (pol a) with primer/templates were examined. Sim... more The interactions of calf thymus DNA polymerase a (pol a) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primerftemplate binding or dNTP polymerization (Kmi Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol a (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the ability of pol a to discriminate against nucleotide analogs, it did not compromise the ability of pol a to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol a strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol a.

Research paper thumbnail of Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase α

Journal of Biological Chemistry

Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templ... more Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templates of defined sequence. Primase seldom misincorporated N T P s during initiation of a new primer (Le. polymerization of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accurate nucleotide-polymerizing enzyme known. In some cases primase discriminated against incorrect NTPs by less than a factor of 100. After primase incorporated a noncognate nucleotide into the primer, the next correct NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing multiple mismatches. Generation of a correctly base-paired primer-template negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After primase synthesized a primer containing multiple mismatches, the primer was transferred to the polymerase a active site via an intramolecular mechanism. Importantly, polymerase a readily elongated this primer if dNTPs were present. These data are discussed with respect to the question of why primase is required for DNA replication.

Research paper thumbnail of 3'-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

Glycoconjugate journal, 1999

In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma ... more In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these re...

Research paper thumbnail of Herpes Simplex Virus 1 Primase Employs Watson−Crick Hydrogen Bonding To Identify Cognate Nucleoside Triphosphates †

Biochemistry, 2005

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by th... more We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.

Research paper thumbnail of Interactions of calf thymus DNA polymerase α with primer/templates

Nucleic Acids Research, 1995

Page 1. © 1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 20 4109-4115 In... more Page 1. © 1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 20 4109-4115 Interactions of calf thymus DNA polymerase a with primer/templates Harry C. Thompson, Robert J. Sheaff* and Robert D. Kuchta* ...

Research paper thumbnail of Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation

Journal of Virology, 2011

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of... more The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5 to 3 helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.

Research paper thumbnail of Inhibition of UDP- N -Acetylglucosamine Import into Golgi Membranes by Nucleoside Monophosphates †

Journal of Medicinal Chemistry, 1996

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucle... more The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5′-phosphate. The enzyme is extremely tolerant to changes at the 3′-position, and the presence of 3′-F actually increases binding of the NMP to the enzyme. At the 2′-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Research paper thumbnail of Detection of adamantane-resistant influenza on a microarray

Journal of Clinical Virology, 2008

A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza the... more A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions.

Research paper thumbnail of Evaluation of MChip with Historic Subtype H1N1 Influenza A Viruses, Including the 1918 "Spanish Flu" Strain

Journal of Clinical Microbiology, 2007

Research paper thumbnail of Fe-S centers in lactyl-CoA dehydratase

Biochemistry, Nov 1, 1986

Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of A... more Lactyl-CoA dehydratase consists of two enzymes, E1 and E2, and requires catalytic quantities of ATP for activity [Kuchta, R. D., & Abeles, R. H. (1985) J. Biol. Chem. 260, 13181-13189]. In contrast to E1, which contains no Fe, E2 contains 8.20 +/- 0.04 mol of Fe/mol of E2, one of which can be removed by 1,10-phenanthroline. E2 also contains 7.33 +/- 0.68 mol of inorganic sulfur/mol of E2, indicating that at least seven of the Fe atoms are present as Fe-S clusters. E1 and E2 contain less than 0.14 mol of Cu, Co, Zn, Mn, and Ni/mol of E1 or E2. Both reduced and oxidized E1 are EPR silent over a 10,000-G scan range at 4 K, while two signals in E2 are observable at 4 K. Identical spectra were obtained with E2 containing either seven or eight Fe atoms, and both signals were only observable at T less than 30 K. Signal 1 has axial symmetry with g = 2.0232 and g = 2.0006. Signal 2 is orthorhombic with g1 = 1.982, g2 = 1.995, and g3 = 2.019. Computer simulation of these spectra with a S = 1/2 spin Hamiltonian was used to extract the g matrices. The intensity of both signals decreases when E2 is reduced with Na2S2O4. We propose that signal 1 is due to an unusual [4Fe-4S] cluster and signal 2 to a [3Fe-3/4S] cluster. Addition of either acrylyl-CoA or lactyl-CoA dramatically alters signal 2.(ABSTRACT TRUNCATED AT 250 WORDS)

Research paper thumbnail of Mechanism of DNA replication fidelity for three mutants of DNA polymerase I: Klenow fragment KF(exo+), KF(polA5), and KF(exo-)

Biochemistry Usa, Mar 1, 1991

Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleo... more Inhibition of the pre-steady-state burst of nucleotide incorporation by a single incorrect nucleotide (nucleotide discrimination) was measured with the Klenow fragment of D N A polymerase I [KF(exo+)].

Research paper thumbnail of Interactions of DNA with Human DNA Primase Monitored with Photoactivatable Cross-Linking Agents: Implications for the Role of the p58 Subunit †

Biochemistry Usa, Oct 1, 1999

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the prim... more Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.

Research paper thumbnail of Dna array analysis as a diagnostic for current and emerging strains of influenza

Research paper thumbnail of Photoinitiator Nucleotide for Quantifying Nucleic Acid Hybridization

Biomacromolecules, Mar 1, 2010

This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sens... more This first report of a photoinitiator-nucleotide conjugate demonstrates a novel approach for sensitive, rapid and visual detection of DNA hybridization events. This approach holds potential for various DNA labeling schemes and for applications benefiting from selective DNA-based polymerization initiators. Here, we demonstrate covalent, enzymatic incorporation of an eosin-photoinitiator 2′deoxyuridine-5′-triphosphate (EITC-dUTP) conjugate into surface-immobilized DNA hybrids. Subsequent radical chain photoinitiation from these sites using an acrylamide/bis-acrylamide formulation yields a dynamic detection range between 500pM and 50nM of DNA target. Increasing EITC-nucleotide surface densities leads to an increase in surface-based polymer film heights until achieving a film height plateau of 280nm ±20nm at 610 ±70 EITC-nucleotides/μm 2 . Film heights of 10-20 nm were obtained from eosin surface densities of approximately 20 EITC-nucleotides/μm 2 while below the detection limit of ~10 EITC-nucleotides/μm 2 , no detectable films were formed. This unique threshold behavior is utilized for instrument-free, visual quantification of target DNA concentration ranges.

Research paper thumbnail of Detection of Antiviral Resistance in Influenza a Using Dna Microarray

Research paper thumbnail of Herpes Simplex Virus-1 Helicase−Primase: Roles of Each Subunit in DNA Binding and Phosphodiester Bond Formation

Biochemistry Usa, Sep 1, 2009

The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and ... more The helicase-primase complex from herpes simplex virus-1 contains three subunits, UL5, UL52, and UL8. We generated each of the potential two subunit complexes, UL5/UL52, UL5/UL8, and UL52/ UL8, and used a series of kinetic and photocrosslinking studies to provide further insights into the roles of each subunit in DNA binding and primer synthesis. UL8 increases the rate of primer synthesis by UL5/UL52 by increasing the rate of primer initiation (2 NTPs → pppNpN), the rate-limiting step in primer synthesis. The UL5/UL8 complex lacked any detectable catalytic activity (DNA dependent ATPase, primase, or RNA polymerase using a RNA primer:template and NTPs as substrates), but could still bind DNA, indicating that UL52 must provide some key amino acids needed for helicase function. The UL52/UL8 complex lacked detectable DNA dependent ATPase activity and could not synthesize primers on single-stranded DNA. However, it exhibited robust RNA polymerase activity using a RNA primer:template and NTPs as substrate. Thus, UL52 must contain the entire primase active site needed for phosphodiester bond formation, while UL5 minimally contributes amino acids needed for initiation of primer synthesis. Photocrosslinking experiments using single-stranded templates containing 5-iodouracil either before, in, or after the canonical 3′-GPyPy (Py = T or C) initiation site for primer synthesis showed that only UL5 crosslinked to the DNA. This occurred for both the UL5/UL52 and UL5/UL52/UL8 complexes, and whether or not the reactions contained NTPs. Photocrosslinking of a RNA primer:template, the product of primer synthesis, containing 5iodouracil in the template generated the same apparent crosslinked species.

Research paper thumbnail of Comparison of the MChip to Viral Culture, Reverse Transcription-PCR, and the QuickVue Influenza AB Test for Rapid Diagnosis of Influenza

The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blin... more The performance of a diagnostic microarray (the MChip assay) for influenza was compared in a blind study to that of viral culture, reverse transcription (RT)-PCR, and the QuickVue Influenza A؉B test. The patient sample data set was composed of 102 respiratory secretion specimens collected between 29 December 2005 and 2 February 2006 at Scott & White Hospital and Clinic in Temple, Texas. Samples were collected from a wide range of age groups by using direct collection, nasal/nasopharyngeal swabs, or nasopharyngeal aspiration. Viral culture and the QuickVue assay were performed at the Texas site at the time of collection. Aliquots for each sample, identified only by study numbers, were provided to the University of Colorado and Vanderbilt University teams for blinded analysis. When referenced to viral culture, the MChip exhibited a clinical sensitivity of 98% and a clinical specificity of 98%. When referenced to RT-PCR, the MChip assay exhibited a clinical sensitivity of 92% and a clinical specificity of 98%. While the MChip assay currently requires 7 to 8 h to complete the analysis, a significant advantage of the test for influenza virus-positive samples is simultaneous detection and full subtype identification for the two subtypes currently circulating in humans (A/H3N2 and A/H1N1) and avian (A/H5N1) viruses.

Research paper thumbnail of Mechanism of Primer Synthesis by the Herpes Simplex Virus 1 Helicase-Primase �

Biochemistry Usa, Mar 1, 2004

We utilized templates of defined sequence to investigate the mechanism of primer synthesis by her... more We utilized templates of defined sequence to investigate the mechanism of primer synthesis by herpes simplex virus 1 helicase-primase. Under steady-state conditions, the rate of primer synthesis and the size distribution of products remained constant with time, suggesting that the rate-limiting step(s) of primer synthesis occur(s) during primer initiation (at or before the formation of the pppNpN dinucleotide). Consistent with this idea, increasing the concentration of NTPs required for dinucleotide synthesis increased the rate of primer synthesis, whereas increasing the concentration of NTPs not involved in dinucleotide synthesis inhibited primer synthesis. Due to these effects on primer initiation, varying the NTP concentration could affect start site selection on templates containing multiple G-pyr-pyr initiation sites. Increasing the NTP concentration also increased the processivity of primase. However, even at very high concentrations of NTPs, elongation of the dinucleotide into longer products remained relatively inefficient. Primase did not readily elongate preexisting primers under conditions where free template was present in large excess of enzyme. However, if template concentrations were lowered such that primase synthesized primers on all or most of the template present in the reaction, then primase would elongate previously synthesized primers.

Research paper thumbnail of The Technology Transfer Awards honor faculty, inventors, business advisors and entrepreneurs who demonstrate best practices in the technology transfer cycle

Research paper thumbnail of Hall, E. T., Yan, J. P., Melancon, P. & Kuchta, R. D. 3'-Azido-3'-deoxythymidine potently inhibits protein glycosylation. A novel mechanism for AZT cytotoxicity. J. Biol. Chem. 269, 14355-1438

Journal of Biological Chemistry

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatm... more 3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (W infection. Unfor-tunateIy, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythpidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This i~i b i t i o n results from direct competition with several pyrimidinesugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve tl-azido-3"deoxythpidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects, thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.

Research paper thumbnail of Interactions of calf thymus DNA polymerase alpha with primer/templates

Nucleic Acids Research

The interactions of calf thymus DNA polymerase a (pol a) with primer/templates were examined. Sim... more The interactions of calf thymus DNA polymerase a (pol a) with primer/templates were examined. Simply changing the primer from DNA to RNA had little effect on primerftemplate binding or dNTP polymerization (Kmi Vmax and processivity). Surprisingly, however, adding a 5'-triphosphate to the primer greatly changed its interactions with pol a (binding, Vmax and Km and processivity). While changing the primer from DNA to RNA greatly altered the ability of pol a to discriminate against nucleotide analogs, it did not compromise the ability of pol a to discriminate against non-cognate dNTPs. Thus the nature of the primer appears to affect 'sugar fidelity', without altering 'base fidelity'. DNase protection assays showed that pol a strongly protected 9 nt of the primer strand, 13 nt of the duplex template strand and 14 nt of the single-stranded template from hydrolysis by DNase I and weakly protected several bases outside this core region. This large DNA binding domain may account for the ability of a 5'-triphosphate on RNA primers to alter the catalytic properties of pol a.

Research paper thumbnail of Misincorporation of nucleotides by calf thymus DNA primase and elongation of primers containing multiple noncognate nucleotides by DNA polymerase α

Journal of Biological Chemistry

Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templ... more Misincorporation of nucleotides by calf thymus DNA primase was examined using synthetic DNA templates of defined sequence. Primase seldom misincorporated N T P s during initiation of a new primer (Le. polymerization of two NTPs to generate the dinucleotide). Following dinucleotide formation, however, primase readily misincorporated NTPs. Although the rate of misincorporation varied according to both the identity of the mismatch and the template sequence, primase is by far the least accurate nucleotide-polymerizing enzyme known. In some cases primase discriminated against incorrect NTPs by less than a factor of 100. After primase incorporated a noncognate nucleotide into the primer, the next correct NTP was readily added. Remarkably, primase could also polymerize consecutive noncognate nucleotides and generate primers containing multiple mismatches. Generation of a correctly base-paired primer-template negatively regulated further primer synthesis; however, generation of a primer-template containing multiple mismatches did not. After primase synthesized a primer containing multiple mismatches, the primer was transferred to the polymerase a active site via an intramolecular mechanism. Importantly, polymerase a readily elongated this primer if dNTPs were present. These data are discussed with respect to the question of why primase is required for DNA replication.

Research paper thumbnail of 3'-Azidothymidine significantly alters glycosphingolipid synthesis in melanoma cells and decreases the shedding of gangliosides

Glycoconjugate journal, 1999

In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma ... more In this report, we establish that 3'-azido-3'-deoxythymidine (AZT) treatment of melanoma cells greatly alters the pattern of glycosphingolipid biosynthesis. In SK-MEL-30 cells, synthesis of the gangliosides GM3 and GD3 was significantly inhibited (60% and 50% of control, respectively) and the production of their precursor, lactosylceramide, was stimulated by 2.5-fold. Control experiments established that phospholipid synthesis was not affected by AZT treatment, consistent with AZT treatment only affecting lipid biosynthetic reactions that involve glycosylation. Likely as a consequence of decreased rates of ganglioside synthesis, AZT treatment of SK-MEL-30 cells also significantly suppressed the amount of gangliosides shed from the membranes of these cells. Since shedding of gangliosides has been proposed to allow melanoma cells to avoid destruction by the immune system and alterations of glycosphingolipid levels are likely important for the malignant cell phenotype, these re...

Research paper thumbnail of Herpes Simplex Virus 1 Primase Employs Watson−Crick Hydrogen Bonding To Identify Cognate Nucleoside Triphosphates †

Biochemistry, 2005

We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by th... more We utilized NTP analogues containing modified bases to probe the mechanism of NTP selection by the primase activity of the herpes simplex virus 1 helicase-primase complex. Primase readily bound NTP analogues of varying base shape, hydrophobicity, and hydrogen-bonding capacity. Remarkably, primase strongly discriminated against incorporating virtually all of the analogues, even though this enzyme misincorporates natural NTPs at frequencies as high as 1 in 7. This included analogues with bases much more hydrophobic than a natural base (e.g., 4- and 7-trifluoromethylbenzimidazole), a base of similar hydrophobicity as a natural base but with the Watson-Crick hydrogen-bonding groups in unusual positions (7-beta-d-guanine), bases shaped almost identically to the natural bases (4-aminobenzimidazole and 4,6-difluorobenzimidazole), bases shaped very differently than a natural base (e.g., 5- and 6-trifluoromethylbenzimidazole), and bases capable of forming just one Watson-Crick hydrogen bond with the template base (purine and 4-aminobenzimidazole). The only analogues that primase readily polymerized into primers (ITP and 3-deaza-ATP) were those capable of forming Watson-Crick hydrogen bonds with the template base. Thus, herpes primase appears to require the formation of Watson-Crick hydrogen bonds in order to efficiently polymerize a NTP. In contrast to primase's narrow specificity for NTP analogues, the DNA-dependent NTPase activity associated with the herpes primase-helicase complex exhibited very little specificity with respect to NTPs containing unnatural bases. The implications of these results with respect to the mechanism of the helicase-primase and current fidelity models are discussed.

Research paper thumbnail of Interactions of calf thymus DNA polymerase α with primer/templates

Nucleic Acids Research, 1995

Page 1. © 1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 20 4109-4115 In... more Page 1. © 1995 Oxford University Press Nucleic Acids Research, 1995, Vol. 23, No. 20 4109-4115 Interactions of calf thymus DNA polymerase a with primer/templates Harry C. Thompson, Robert J. Sheaff* and Robert D. Kuchta* ...

Research paper thumbnail of Herpes Simplex Virus Type 1 Helicase-Primase: DNA Binding and Consequent Protein Oligomerization and Primase Activation

Journal of Virology, 2011

The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of... more The heterotrimeric helicase-primase complex of herpes simplex virus type I (HSV-1), consisting of UL5, UL8, and UL52, possesses 5 to 3 helicase, single-stranded DNA (ssDNA)-dependent ATPase, primase, and DNA binding activities. In this study we confirm that the UL5-UL8-UL52 complex has higher affinity for forked DNA than for ssDNA and fails to bind to fully annealed double-stranded DNA substrates. In addition, we show that a single-stranded overhang of greater than 6 nucleotides is required for efficient enzyme loading and unwinding. Electrophoretic mobility shift assays and surface plasmon resonance analysis provide additional quantitative information about how the UL5-UL8-UL52 complex associates with the replication fork. Although it has previously been reported that in the absence of DNA and nucleoside triphosphates the UL5-UL8-UL52 complex exists as a monomer in solution, we now present evidence that in the presence of forked DNA and AMP-PNP, higher-order complexes can form. Electrophoretic mobility shift assays reveal two discrete complexes with different mobilities only when helicase-primase is bound to DNA containing a single-stranded region, and surface plasmon resonance analysis confirms larger amounts of the complex bound to forked substrates than to single-overhang substrates. Furthermore, we show that primase activity exhibits a cooperative dependence on protein concentration while ATPase and helicase activities do not. Taken together, these data suggest that the primase activity of the helicase-primase requires formation of a dimer or higher-order structure while ATPase activity does not. Importantly, this provides a simple mechanism for generating a two-polymerase replisome at the replication fork.

Research paper thumbnail of Inhibition of UDP- N -Acetylglucosamine Import into Golgi Membranes by Nucleoside Monophosphates †

Journal of Medicinal Chemistry, 1996

The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucle... more The specificity of the UDP-N-acetylglucosamine (UDP-GlcNAc) translocator for the binding of nucleoside monophosphates (NMPs) and nucleotide-sugars was examined in order to develop a quantitative understanding of how this enzyme recognizes its substrates and to provide a framework for development of novel drugs that target glycosylation. Competition studies reveal that tight binding requires a complete ribose ring and a 5′-phosphate. The enzyme is extremely tolerant to changes at the 3′-position, and the presence of 3′-F actually increases binding of the NMP to the enzyme. At the 2′-position, substitutions in the ribo configuration are well tolerated, although these same substitutions greatly diminish binding when present in the ara configuration. For the base, size appears to be the key feature for discrimination. The enzyme tolerates changing the C-4 oxygen of uridine to an amino group as well as substituting groups containing one or two carbons at C-5. However, substitution of groups containing three carbons at C-5, or exchange of the pyrimidine for a purine, greatly weakens binding to the translocator. Comparison of various UDP-sugars reveals that the UDP-GlcNAc translocator has lower affinity for UDP-N-acetylgalactosamine and UDP-glucose than for its cognate substrate and therefore indicates that this translocator requires both proper stereochemistry at C-4 and an aminoacetyl group at C-2. The impact of these observations on the design of more powerful nucleoside-based inhibitors of nucleotide-sugar import is discussed.

Research paper thumbnail of Detection of adamantane-resistant influenza on a microarray

Journal of Clinical Virology, 2008

A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza the... more A has the ability to rapidly mutate and become resistant to the commonly prescribed influenza therapeutics, thereby complicating treatment decisions.

Research paper thumbnail of Evaluation of MChip with Historic Subtype H1N1 Influenza A Viruses, Including the 1918 "Spanish Flu" Strain

Journal of Clinical Microbiology, 2007