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Corey Devin Anderson - One of the best experts on this subject based on the ideXlab platform.
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utility of a set of microsatellite Primers developed for the massasauga rattlesnake sistrurus catenatus for population genetic studies of the timber rattlesnake crotalus horridus
Molecular Ecology Notes, 2006Co-Authors: Corey Devin AndersonAbstract:I tested six microsatellite DNA Primer pairs developed for the massasauga rattlesnake (Sistrurus catenatus) on a sample population of the timber rattlesnake (Crotalus horridus). It had been speculated in a previous publication that cross-species amplification would not be worthwhile across the two rattlesnake genera. However, for this Primer set (the only one currently published for the genus Sistrurus), successful amplification at each locus was accomplished for all loci with an annealing temperature of 57 °C and locus-specific buffer conditions. Each locus was polymorphic, with the number of alleles per locus ranging from two to 12. Significant heterozygote deficits were detected for three loci (Scu01, Scu05 and Scu07). For Scu01, all individuals were homozygous for the same allele except one female who was homozygous for a different allele. This same female was also homozygous for a rare allele at Scu07. When this female was removed from the data set, the number of observed heterozygotes at Scu01 and Scu07 did not differ significantly from random expectations. However, a large heterozygote deficit persisted at Scu05 (despite subsampling), suggesting that this locus may not be useful for population genetic studies of timber rattlesnakes. Despite some limitations, this set of Primers may be a useful complement to those already developed for the genus Crotalus. Moreover, the results of this study seem to provide new justification for further studies of cross-species amplification of microsatellite loci across the two rattlesnake genera.
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utility of a set of microsatellite Primers developed for the massasauga rattlesnake sistrurus catenatus for population genetic studies of the timber rattlesnake crotalus horridus
Molecular Ecology Notes, 2006Co-Authors: Corey Devin AndersonAbstract:I tested six microsatellite DNA Primer pairs developed for the massasauga rattlesnake (Sistrurus catenatus) on a sample population of the timber rattlesnake (Crotalus horridus). It had been speculated in a previous publication that cross-species amplification would not be worthwhile across the two rattlesnake genera. However, for this Primer set (the only one currently published for the genus Sistrurus), successful amplification at each locus was accomplished for all loci with an annealing temperature of 57 °C and locus-specific buffer conditions. Each locus was polymorphic, with the number of alleles per locus ranging from two to 12. Significant heterozygote deficits were detected for three loci (Scu01, Scu05 and Scu07). For Scu01, all individuals were homozygous for the same allele except one female who was homozygous for a different allele. This same female was also homozygous for a rare allele at Scu07. When this female was removed from the data set, the number of observed heterozygotes at Scu01 and Scu07 did not differ significantly from random expectations. However, a large heterozygote deficit persisted at Scu05 (despite subsampling), suggesting that this locus may not be useful for population genetic studies of timber rattlesnakes. Despite some limitations, this set of Primers may be a useful complement to those already developed for the genus Crotalus. Moreover, the results of this study seem to provide new justification for further studies of cross-species amplification of microsatellite loci across the two rattlesnake genera.
Ulrich Hubscher - One of the best experts on this subject based on the ideXlab platform.
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Okazaki fragment processing: Modulation of the strand displacement activity of DNA polymerase δ by the concerted action of replication protein A, proliferating cell nuclear antigen, and flap endonuclease-1
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Giovanni Maga, Giuseppe Villani, Vanessa Tillement, Manuel Stucki, Giada A. Locatelli, Isabelle Frouin, Silvio Spadari, Ulrich HubscherAbstract:DNA polymerase (pol) delta is essential for both leading and lagging strand DNA synthesis during chromosomal replication in eukaryotes. Pol delta has been implicated in the Okazaki fragment maturation process for the extension of the newly synthesized fragment and for the displacement of the RNA/DNA segment of the preexisting downstream fragment generating an intermediate flap structure that is the target for the DNA2 and flap endonuclease-1 (Fen 1) endonucleases. Using a single-stranded minicircular template with an annealed RNA/DNA Primer, we could measure strand displacement by pol delta coupled to DNA synthesis. Our results suggested that pol delta alone can displace up to 72 nucleotides while synthesizing through a double-stranded DNA region in a distributive manner. Proliferating cell nuclear antigen (PCNA) reduced the template dissociation rate of pol delta, thus increasing the processivity of both synthesis and strand displacement, whereas replication protein A (RP-A) limited the size of the displaced fragment down to 20-30 nucleotides, by generating a "locked" flap DNA structure, which was a substrate for processing of the displaced fragment by Fen 1 into a ligatable product. Our data support a model for Okazaki fragment processing where the strand displacement activity of DNA polymerase delta is modulated by the concerted action of PCNA, RP-A and Fen 1.
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DNA polymerase switching ii replication factor c abrogates Primer synthesis by DNA polymerase alpha at a critical length
Journal of Molecular Biology, 2000Co-Authors: Romina Mossi, Robert Keller, Elena Ferrari, Ulrich HubscherAbstract:Abstract A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA Primer by DNA polymerase α/primase to the pro?cessive elongation by DNA polymerase δ. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase α were investigated. We show that RF-C stalls DNA polymerase α after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se . This suggested that RF-C and the length of the Primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5′ recessed ends and double-stranded DNA over 3′ ends. Finally PCNA can be loaded onto a DNA template carrying a RNA Primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA Primer, influences Primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.
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DNA polymerase switching ii replication factor c abrogates Primer synthesis by DNA polymerase alpha at a critical length
Journal of Molecular Biology, 2000Co-Authors: Romina Mossi, Robert Keller, Elena Ferrari, Ulrich HubscherAbstract:A crucial event in DNA replication is the polymerase switch from the synthesis of a short RNA/DNA Primer by DNA polymerase alpha/primase to the pro?cessive elongation by DNA polymerase delta. In order to shed light on the role of replication factor C (RF-C) in this process, the effects of RF-C on DNA polymerase alpha were investigated. We show that RF-C stalls DNA polymerase alpha after synthesis of approximately 30 nucleotides, while not inhibiting the polymerase activity per se. This suggested that RF-C and the length of the Primer may be two important factors contributing to the polymerase switch. Furthermore the DNA binding properties of RF-C were tested. Band shift experiments indicated that RF-C has a preference for 5' recessed ends and double-stranded DNA over 3' ends. Finally PCNA can be loaded onto a DNA template carrying a RNA Primer, suggesting that a DNA moiety is not necessarily required for the loading of the clamp. Thus we propose a model where RF-C, upon binding to the RNA/DNA Primer, influences Primer synthesis and sets the conditions for a polymerase switch after recruiting PCNA to DNA.
Robert A Bambara - One of the best experts on this subject based on the ideXlab platform.
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flap endonuclease disengages DNA2 helicase nuclease from okazaki fragment flaps
Journal of Biological Chemistry, 2006Co-Authors: Jason A. Stewart, Judith L. Campbell, Robert A BambaraAbstract:Okazaki fragments contain an initiator RNA/DNA Primer that must be removed before the fragments are joined. In eukaryotes, the Primer region is raised into a flap by the strand displacement activity of DNA polymerase {delta}. The DNA2 helicase/nuclease and then flap endonuclease 1 (FEN1) are proposed to act sequentially in flap removal. DNA2 and FEN1 both employ a tracking mechanism to enter the flap 5' end and move toward the base for cleavage. In the current model, DNA2 must enter first, but FEN1 makes the final cut at the flap base, raising the issue of how FEN1 passes the DNA2. To address this, nuclease-inactive DNA2 was incubated with a DNA flap substrate and found to bind with high affinity. FEN1 was then added, and surprisingly, there was little inhibition of FEN1 cleavage activity. FEN1 was later shown, by gel shift analysis, to remove the wild type DNA2 from the flap. RNA can be cleaved by FEN1 but not by DNA2. Pre-bound wild type DNA2 was shown to bind an RNA flap but not inhibit subsequent FEN1 cleavage. These results indicate that there is a novel interaction between the two proteins in which FEN1 disengages the DNA2 tracking mechanism. This interaction is consistent with the idea that the two proteins have evolved a special ability to cooperate in Okazaki fragment processing.
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Reconstituted Okazaki fragment processing indicates two pathways of Primer removal.
Journal of Biological Chemistry, 2006Co-Authors: Marie L. Rossi, Robert A BambaraAbstract:Abstract Eukaryotic Okazaki fragments are initiated by an RNA/DNA Primer and extended by DNA polymerase δ (pol δ) and the replication clamp proliferating cell nuclear antigen (PCNA). Joining of the fragments by DNA ligase I to generate the continuous double-stranded DNA requires complete removal of the RNA/DNA Primer. Pol δ extends the upstream Okazaki fragment and displaces the downstream RNA/DNA Primer into a flap removed by nuclease cleavage. One proposed pathway for flap removal involves pol δ displacement of long flaps, coating of those flaps by replication protein A (RPA), and sequential cleavage of the flap by DNA2 nuclease followed by flap endonuclease 1 (FEN1). A second pathway involves reiterative single nucleotide or short oligonucleotide displacement by pol δ and cleavage by FEN1. We measured the length of FEN1 cleavage products on flaps strand-displaced by pol δ in an oligonucleotide system reconstituted with Saccharomyces cerevisiae proteins. Results showed that in the presence of PCNA and FEN1, pol δ displacement synthesis favors formation and cleavage of primarily short flaps, up to eight nucleotides in length; still, a portion of flaps grows to 20–30 nucleotides. The proportion of long flaps can be altered by mutations in the relevant proteins, sequence changes in the DNA, and reaction conditions. These results suggest that FEN1 is sufficient to remove a majority of Okazaki fragment Primers. However, some flaps become long and require the two-nuclease pathway. It appears that both pathways, operating in parallel, are required for processing of all flaps.
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On the Roles of Saccharomyces cerevisiae DNA2p and Flap Endonuclease 1 in Okazaki Fragment Processing
Journal of Biological Chemistry, 2004Co-Authors: Hui-i Kao, Judith L. Campbell, Janaki Veeraraghavan, Piotr Polaczek, Robert A BambaraAbstract:Abstract Short DNA segments designated Okazaki fragments are intermediates in eukaryotic DNA replication. Each contains an initiator RNA/DNA Primer (iRNA/DNA), which is converted into a 5′-flap and then removed prior to fragment joining. In one model for this process, the flap endonuclease 1 (FEN1) removes the iRNA. In the other, the single-stranded binding protein, replication protein A (RPA), coats the flap, inhibits FEN1, but stimulates cleavage by the DNA2p helicase/nuclease. RPA dissociates from the resultant short flap, allowing FEN1 cleavage. To determine the most likely process, we analyzed cleavage of short and long 5′-flaps. FEN1 cleaves 10-nucleotide fixed or equilibrating flaps in an efficient reaction, insensitive to even high levels of RPA or DNA2p. On 30-nucleotide fixed or equilibrating flaps, RPA partially inhibits FEN1. CTG flaps can form foldback structures and were inhibitory to both nucleases, however, addition of a dT12 to the 5′-end of a CTG flap allowed DNA2p cleavage. The presence of high DNA2p activity, under reaction conditions favoring helicase activity, substantially stimulated FEN1 cleavage of tailed-foldback flaps and also 30-nucleotide unstructured flaps. Our results suggest DNA2p is not used for processing of most flaps. However, DNA2p has a role in a pathway for processing structured flaps, in which it aids FEN1 using both its nuclease and helicase activities.
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nevirapine alters the cleavage specificity of ribonuclease h of human immunodeficiency virus 1 reverse transcriptase
Journal of Biological Chemistry, 1995Co-Authors: Chockalingam Palaniappan, Philip J Fay, Robert A BambaraAbstract:Abstract The action of the dipyridodiazepinone nevirapine (BI-RG-587) on polymerization and RNase H activities of human immunodeficiency virus reverse transcriptase (RT) was examined. Substrates using heteropolymeric DNA Primers hybridized to complementary RNA templates were employed. Challenged assays were performed that allowed measurement of activity of the RT resulting from a single round of binding of RT to substrate. Results demonstrated that nevirapine alters the cleavage specificity of the RNase H. Instead of a primary cleavage approximately 18 nucleotides upstream of the DNA 3′ terminus, multiple cleavages were observed ahead of and behind this site. This indicated that the compound facilitates sliding of the RT away from the DNA Primer terminus allowing cleavage at more sites. The change in specificity occurred whether the Primer terminus was at the end or internal on the template. Experiments with RNA Primers on circular DNA demonstrated a nevirapine-induced stimulation of RNase H activity beyond the increase expected from the change in cleavage specificity. Examination of polymerization showed that the compound decreased both the number of Primers that underwent synthesis and the processive elongation of those Primers. The significance of these results with respect to viral replication and recombination is discussed.
Michael Nassal - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of a functional duck hepatitis b virus replication initiation complex from separate reverse transcriptase domains expressed in escherichia coli
Journal of Virology, 2001Co-Authors: Jurgen Beck, Michael NassalAbstract:Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (ɛ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA Primer that is templated by ɛ and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli , followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of ɛ-dependent DNA Primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepaDNAviral replication initiation mechanism.
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formation of a functional hepatitis b virus replication initiation complex involves a major structural alteration in the rna template
Molecular and Cellular Biology, 1998Co-Authors: Jurgen Beck, Michael NassalAbstract:The DNA genome of a hepatitis B virus is generated by reverse transcription of the RNA pregenome. Replication initiation does not involve a nucleic acid Primer; instead, the hepaDNAvirus P protein binds to the structured RNA encapsidation signal epsilon, from which it copies a short DNA Primer that becomes covalently linked to the enzyme. Using in vitro-translated duck hepatitis B virus (DHBV) P protein, we probed the secondary structure of the protein-bound DHBV epsilon RNA (Depsilon) and observed a marked conformational change compared to free Depsilon RNA. Several initiation-competent mutant RNAs with a different free-state structure were similarly altered, whereas a binding-competent but initiation-deficient variant was not, indicating the importance of the rearrangement for replication initiation and suggesting a mechanistic coupling to encapsidation.
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a bulged region of the hepatitis b virus rna encapsidation signal contains the replication origin for discontinuous first strand DNA synthesis
Journal of Virology, 1996Co-Authors: Michael Nassal, A RiegerAbstract:Human hepatitis B virus (HBV) is a small DNA virus that replicates inside the viral nucleocapsid by reverse transcription of an RNA intermediate. Encapsidation of this RNA pregenome is mediated by the interaction of the viral replication enzyme P with the structured 5'-proximal RNA element epsilon; replication was thought to start in the 3'-proximal direct repeat DR1*. However, recent data obtained with the duck hepatitis B virus indicated a novel, discontinuous mechanism of negative-strand DNA synthesis. Here we demonstrate, using DNA transfection of complete HBV genomes, that the 3'-half of a 6-nucleotide bulge in HBV epsilon whose primary sequence is not important for encapsidation serves as template for a short DNA Primer that is subsequently transferred to DR1*. Apparently, P protein copies any template sequence that does not interfere with epsilon structure; however, altered primary sequences can induce polymerase stuttering, resulting in extended Primers containing more than one equivalent of the template sequence. The importance of the bulged structure is emphasized by the dependence of Primer length on bulge size. Transfer specificity is in part controlled by sequence complementarity. The strategy of using the 5' encapsidation signal as the origin of replication for discontinuous negative-strand DNA synthesis, common to mammalian and avian hepaDNAviruses, suggests the evolutionary origin of hepatitis B viruses to lie between that of modern retroviruses and primitive retroelements like the Mauriceville retroplasmid.
Luis Blanco - One of the best experts on this subject based on the ideXlab platform.
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engineering human primpol into an efficient rna dependent DNA primase polymerase
Nucleic Acids Research, 2017Co-Authors: Ruben Agudo, Maria I Martinezjimenez, Patricia A Calvo, Luis BlancoAbstract:We have developed a straightforward fluorometric assay to measure primase-polymerase activity of human PrimPol (HsPrimPol). The sensitivity of this procedure uncovered a novel RNA-dependent DNA priming-polymerization activity (RdDP) of this enzyme. In an attempt to enhance HsPrimPol RdDP activity, we constructed a smart mutant library guided by prior sequence-function analysis, and tested this library in an adapted screening platform of our fluorometric assay. After screening less than 500 variants, we found a specific HsPrimPol mutant, Y89R, which displays 10-fold higher RdDP activity than the wild-type enzyme. The improvement of RdDP activity in the Y89R variant was due mainly to an increased in the stabilization of the preternary complex (protein:template:incoming nucleotide), a specific step preceding dimer formation. Finally, in support of the biotechnological potential of PrimPol as a DNA Primer maker during reverse transcription, mutant Y89R HsPrimPol rendered up to 17-fold more DNA than with random hexamer Primers.