DNA Polymerase X From African Swine Fever Virus: Quantitative Analysis of the Enzyme–ssDNA Interactions and the Functional Structure of the Complex

Jezewska, Maria J.; Marcinowicz, Agnieszka; Lucius, Aaron L.; Bujalowski, Wlodzimierz

doi:10.1016/j.jmb.2005.10.061

Cited by 22 publications

(125 citation statements)

References 60 publications

(322 reference statements)

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“…The advantage of using the etheno-derivatives of the ssDNA and ssRNA is that their fluorescence depends little upon the polarity of the environment and predominantly reflects the structure of the nucleic acid (23,60). The linear dependence of ⌬F obs over a large range of ⌺v i does not exclude the heterogeneous structure of the total binding site, as is the case of the African swine fever virus polymerase X (28,36). Nevertheless, it indicates that, similar to the African swine fever virus polymerase X, the full-length DENV polymerase forms a single type of complex with the polymer nucleic acid.…”

Section: Discussionmentioning

confidence: 99%

“…In studies described in this work, we use, as a reference lattice, poly(⑀A), and the base specificity of the DENV polymerase has been examined using different ssRNA homopolymers, poly(A), poly(U), and poly(C). At a given titration point, i, the total concentration of the bound protein, P b , is defined as follows (31,32,36,38),…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

“…To overcome the problem of resorting to complex numerical calculations, one can apply a combined application of the generalized McGheevon Hippel model, as defined by Equations 8 and 9, and the combinatorial theory for large ligand binding to a linear, homogeneous lattice (31,32,36,38). In this approach, the ligand binding to the reference fluorescent nucleic acid is described by the generalized McGhee-von Hippel equation.…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

“…Thus, both 30-mers should bind only a single molecule of the polymerase, which greatly simplifies the binding analysis. The reference fluorescent nucleic acid is poly(⑀A) (31,32,36,38). In the presence of the unmodified 30-mer, at a given titration point, i, the total concentration of the bound protein, P b , is defined by an expression analogous to Equation 10, as follows, where (⌺v i ) R and (⌺⍜ i ) S are the total average binding density of the DENV polymerase on the reference poly(⑀A) and the total average degree of binding on the examined unmodified 30-mer, respectively.…”

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

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Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Szymanski

Jezewska

Bujalowski

et al. 2011

Journal of Biological Chemistry

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Fundamental aspects of interactions of the Dengue virus type 3 full-length polymerase with the single-stranded and doublestranded RNA and DNA have been quantitatively addressed. The polymerase exists as a monomer with an elongated shape in solution. In the absence of magnesium, the total site size of the polymerase-ssRNA complex is 26 ؎ 2 nucleotides. In the presence of Mg 2؉ , the site size increases to 29 ؎ 2 nucleotides, indicating that magnesium affects the enzyme global conformation. The enzyme shows a preference for the homopyrimidine ssRNAs. Positive cooperativity in the binding to homopurine ssRNAs indicates that the type of nucleic acid base dramatically affects the enzyme orientation in the complex. Both the intrinsic affinity and the cooperative interactions are accompanied by a net ion release. The polymerase binds the dsDNA with an affinity comparable with the ssRNAs affinity, indicating that the binding site has an open conformation in solution. The lack of detectable dsRNA or dsRNA-DNA hybrid affinities indicates that the entry to the binding site is specific for the sugar-phosphate backbone and/or conformation of the duplex. The dengue virus (DENV)2 is a member of the Flaviviridae family and the etiological agent of dengue fever, a disease affecting worldwide ϳ100 million people each year (1-5). The Flaviviridae family includes such human pathogens as tick-borne encephalitis virus, hepatitis C virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus (1-5). The dengue virus exists in four distinct types, DENV1, -2, -3, and -4, which show ϳ60% genomic sequence identity (1). The dengue fever often develops into the dengue hemorrhagic fever, an acute form of the disease, possibly as a result of sequential infection with different types of the virus. It is characterized by a significant mortality, particularly for individuals with a weakened or undeveloped immune system. The small, 10.7-kbp positive strand RNA genome of the dengue virus encodes only 10 proteins: three structural proteins (capsid, premembrane, and envelope proteins) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 proteins) (3, 6 -8). The entire viral genome is translated as a single polyprotein, subsequently cleaved by viral and cellular proteases into functional entities.The NS5 protein is the viral replicative RNA-dependent RNA polymerase (1, 3, 6 -8). The primary structure of the fulllength polymerase encompasses 900 amino acids (i.e. it is the largest of the nonstructural proteins of the dengue virus) (6 -10). The enzyme is built of two functional domains (6 -10). The N-terminal domain includes the first 263 amino acids. It functions as a methyltransferase (MTase), whose activity is necessary for viral RNA recognition by the host cell translational apparatus. Correspondingly, amino acids ϳ273-906 form the polymerase domain, containing the active site of the RNA synthesis (Fig. 1a) (6 -8). The crystal structures of the isolated N-terminal and polymerase domains have been determined up t...

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Section: Discussionmentioning

confidence: 99%

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

Section: Base Specificity Of Denv Polymerase-ssdna Interactions; Lattmentioning

confidence: 99%

See 2 more Smart Citations

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Szymanski

Jezewska

Bujalowski

et al. 2011

Journal of Biological Chemistry

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“…These include (lack of) amino-terminal pol β-like lyase and DNA binding domains, a unique overall shape and a unique secondary structure in the catalytic subdomain, the lowest reported fidelity of characterized polymerases including an inability to discriminate between G:C and G:G base pairs, and a unique DNA binding mechanism that may affect active-site organization and contribute to low fidelity (Beard and Wilson 2001;Jezewska et al 2006;Maciejewski et al 2001;Sampoli Benitez et al 2006;. These characteristics have led to the suggestion that pol X may act as a mutase or have a role in a mutagenic repair system responsible for generating ASFV genetic heterogeneity.…”

Section: Viral Dna Replicationmentioning

confidence: 99%

African Swine Fever Virus

Tulman

Delhon

et al. 2009

Current Topics in Microbiology and Immunology

135

117

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African swine fever virus (ASFV) is a large, intracytoplasmicallyreplicating DNA arbovirus and the sole member of the family Asfarviridae . It is the etiologic agent of a highly lethal hemorrhagic disease of domestic swine and therefore extensively studied to elucidate the structures, genes, and mechanisms affecting viral replication in the host, virus-host interactions, and viral virulence. Increasingly apparent is the complexity with which ASFV replicates and interacts with the host cell during infection. ASFV encodes novel genes

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Structure and function of 2:1 DNA polymerase·DNA complexes

Tang

Tsai

2008

Journal Cellular Physiology

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DNA polymerases are required for DNA replication and DNA repair in all of the living organisms. Different DNA polymerases are responsible different stages of DNA metabolism, and many of them are multifunctional enzymes. It was generally assumed that the different reactions are catalyzed by the same enzyme molecule. In addition to 1:1 DNA polymerase.DNA complex reported by crystallization studies, 2:1 and higher order DNA polymerase.DNA complexes have been identified in solution studies by various biochemical and biophysical approaches. Further, abundant evidences for the DNA polymerase-DNA interactions in several DNA polymerases suggested that the 2:1 complex represents the more active form. This review describes the current status of this emerging subject and explores their potential in vitro and in vivo functional significance, particularly for the 2:1 complexes of mammalian DNA polymerase beta (Pol beta), the Klenow fragment of E. coli DNA polymerase I (KF), and T4 DNA polymerase.

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DNA Polymerase X From African Swine Fever Virus: Quantitative Analysis of the Enzyme–ssDNA Interactions and the Functional Structure of the Complex

Cited by 22 publications

References 60 publications

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

Full-length Dengue Virus RNA-dependent RNA Polymerase-RNA/DNA Complexes

African Swine Fever Virus

Structure and function of 2:1 DNA polymerase·DNA complexes

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