Chloroacetaldehyde-modified dinucleoside phosphates. Dynamic fluorescence quenching and quenching due to intramolecular complexation

Tolman, Glen L.; Barrio, Jorge R.; Leonard, Nelson J.

doi:10.1021/bi00721a001

Cited by 93 publications

(76 citation statements)

References 41 publications

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“…Because at the applied excitation wavelength ( ex ϭ 325 nm), only ethenoadenosine is excited, the observed increase results from an increase of the quantum yield of the nucleic acid in the complex with the polymerase. The fluorescence of ⑀A is dramatically quenched (8 -12-fold) in etheno-oligomers and polymers as compared with the free AMP (44,45). A dynamic model, in which the motion of ⑀A leads to quenching via intramolecular collision, has been proposed as a predominant mechanism of the observed strong quenching (44).…”

Section: Discussionmentioning

confidence: 99%

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Jezewska

Rajendran

Bujalowski

2001

Journal of Biological Chemistry

135

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Interactions between rat polymerase ␤ (pol ␤) and the template-primer, as well as gapped DNAs, were studied using the quantitative fluorescence titration technique. Stoichiometries of rat pol ␤ complexes with DNA substrates are much higher than stoichiometries predicted by the structures of co-crystals. The data can be understood in the context of the two single-stranded (ss)DNAbinding modes of the enzyme, the (pol ␤ Polymerase ␤ is one of a number of recognized DNA-directed polymerases of the eukaryotic nucleus (1-5). The enzyme has a very specialized function in mammalian cell repair machinery. The activities of pol ␤ 1 have been proposed to include the gap filling synthesis involved in mismatch repair (4, 6 -8), in the repair of monofunctional adducts, UV damaged DNA, and abasic lesions in DNA (9 -14). Thus, a processive "gap fillings" synthesis, observed in vitro, by rat pol ␤ on gapped DNAs, is consistent with the proposed in vivo activities (2, 6 -8). A characteristic feature of rat pol ␤ is a "simplified" repertoire of activities. The enzyme is lacking intrinsic accessory activities, such as 3Ј-or 5Ј-exonuclease, endonuclease, dNMP turnover, and pyrophosphorolysis (1-4, 6, 7). Such limited activities reflect the very specialized function of the polymerase in the DNA metabolism.)The crystal structure of the rat pol ␤ enzyme revealed a typical polymerase fold, a thumb, palm, and fingers, due to its resemblance to the human hand (15-17). However, what distinguishes the pol ␤ structure from other polymerases is the presence of a small 8-kDa domain that is connected with the tip of the fingers through a tether of 14 amino acids (15-17). Solution studies showed that the 8-kDa domain has significant affinity for the single-stranded (ss) DNA, indicating that the domain plays a key role in recognition of this nucleic acid conformation, i.e. it is the template-binding domain (15,18,19,21,22). The active site of the DNA synthesis and the dsDNA affinity were proposed to reside predominantly in the large 31-kDa catalytic domain (15,18,19,23).Recently, we examined interactions of rat and human polymerase ␤ with the ssDNA (20, 24). Our data showed that rat pol ␤ binds the ssDNA in two binding modes which differ in the number of occluded nucleotide residues, the (pol ␤) 16 and (pol ␤) 5 binding mode. The binding modes differ in affinities and abilities to induce conformational changes in the ssDNA (20). Thus, the intrinsic affinity of the enzyme in the (pol ␤) 16 binding mode is approximately an order of magnitude higher than the affinity in the (pol ␤) 5 binding mode. On the other hand, when bound in the (pol ␤) 5 binding mode, the enzyme induces much more profound structural changes in the ssDNA, suggesting strong base-base separation and DNA immobilization in the complex (20). The obtained results also indicate that in the (pol ␤) 16 binding mode, both the 8-and 31-kDa domain of the enzyme, are involved in interactions with the ssDNA. In the (pol ␤) 5 binding mode, the 8-kDa domain predominately is engaged in interact...

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Section: Discussionmentioning

confidence: 99%

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Jezewska

Rajendran

Bujalowski

2001

Journal of Biological Chemistry

135

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“…The etheno-derivative of A(pA) 19 , A(pA) 19 , was obtained by modification of A(pA) 19 with chloroacetaldehyde. This modification goes to completion and provides a fluorescent derivative of the nucleic acid (29,34,35).…”

Section: Methodsmentioning

confidence: 99%

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

Bujalowski

Choi

2017

J Virol

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The process of RNA replication by dengue virus is still not completely understood despite the significant progress made in the last few years. Stem-loop A (SLA), a part of the viral 5= untranslated region (UTR), is critical for the initiation of dengue virus replication, but quantitative analysis of the interactions between the dengue virus polymerase NS5 and SLA in solution has not been performed. Here, we examine how solution conditions affect the size and shape of SLA and the formation of the NS5-SLA complex. We show that dengue virus NS5 binds SLA with a 1:1 stoichiometry and that the association reaction is primarily entropy driven. We also observe that the NS5-SLA interaction is influenced by the magnesium concentration in a complex manner. Binding is optimal with 1 mM MgCl 2 but decreases with both lower and higher magnesium concentrations. Additionally, data from a competition assay between SLA and single-stranded RNA (ssRNA) indicate that SLA competes with ssRNA for the same binding site on the NS5 polymerase. SLA 70 and SLA 80 , which contain the first 70 and 80 nucleotides (nt), respectively, bind NS5 with similar binding affinities. Dengue virus NS5 also binds SLAs from different serotypes, indicating that NS5 recognizes the overall shape of SLA as well as specific nucleotides.IMPORTANCE Dengue virus is an important human pathogen responsible for dengue hemorrhagic fever, whose global incidence has increased dramatically over the last several decades. Despite the clear medical importance of dengue virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. In particular, stem-loop A (SLA) and stem-loop B (SLB) located in the 5= untranslated region (UTR) are critical for binding the viral polymerase NS5 to initiate minus-strand RNA synthesis. However, little is known regarding the kinetic and thermodynamic parameters driving these interactions. Here, we quantitatively examine the energetics of intrinsic affinities, characterize the stoichiometry of the complex of NS5 and SLA, and determine how solution conditions such as magnesium and sodium concentrations and temperature influence NS5-SLA interactions in solution. Quantitatively characterizing dengue virus NS5-SLA interactions will facilitate the design and assessment of antiviral therapeutics that target this essential step of the dengue virus life cycle. KEYWORDS NS5, RNA polymerase, dengue virus, stem-loop A D engue virus (DENV) is a positive-sense, single-stranded RNA (ssRNA) virus and is a member of the Flavivirus genus within the Flaviviridae family. DENV causes a wide range of diseases in humans, ranging from self-limiting dengue fever to life-threatening dengue hemorrhagic fever and dengue shock syndrome (1-5). Recent estimates indicate that more than 300 million dengue virus infections occur each year, 96 million of which clinically manifest. Currently, it is estimated that 3.9 billion people living in 128 countries are at risk of infection by DENV (6, 7). Four ...

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“…In that connection, from a plot of In Ka against temperature for Ade9-C -Ind3, AH0 --8.1 + 0.9 kcal/mol and ASO =-26.6 + 3.0 e.u. Equivalent or greater complexation is observed in other systems in which e-adenosine replaced adenosine (23,31,47 (1973)(1974) from Eli Lilly and Co.…”

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confidence: 87%

Dynamic and static quenching of 1,N6-ethenoadenine fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3-(indol-3-yl) propyl) adenine.

Gruber¹,

Leonard²

1975

Proc. Natl. Acad. Sci. U.S.A.

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For nicotinamide 1,N6-ethenoadenine dinucleotide (ENAD+), the fluorescent analog of NAD+, in neutral aqueous solution the quantum yield has been determined to be 0.028 and the fluorescent lifetime, 2.1 nsec. Simultaneous determination of quantum yields and lifetimes of eNAD+ and of the "half molecule" EAMP allows the calculation of the percentage of stacked and open conformations of the dinucleotide. At 250 in neutral aqueous solution there is 45 I 5% of stacked forms. The value of the fluorescent lifetime observed for ENAD+ is sensitive to fluorescent impurities, especially those containing the E-adenosine moiety, and a purification procedure using high performance liquid chromatography was devised to obtain fluorescently homogeneous preparations.In order to study the effect on e-adenosine fluorescence caused by the possible close proximity of a tryptophan in a polypeptide chain or protein, we have prepared 1,N6-etheno- These studies relate to intramolecular stacking interactions within the ENAD+ molecule and set a probable upper limit for the interactions within the NAD+ molecule itself. It is also important to delineate possible interactions between the adenosine moiety of various coenzymes and the individual amino acids, e.g., tryptophan, of various proteins. To do so on a simplified level we synthesized 1,N6-etheno-9-[3- (indol-3-yl)propyl]adenine (EAde9-Cs-Ind3) by reaction of 9-[3-(indol-3-yl)propyl]adenine (Ade9-C3-Ind3) (25, 26) with chloroacetaldehyde, thus providing a model in which indole is used as a neutral substitute for tryptophan. From simultaneous measurement of the fluorescence lifetime and the quantum yield of EAde9-C3-Ind3 relative to those of the "half molecules" 1,N6-etheno-9-propyladenine (EAde9-C3) and 3-propylindole (Ind3-Cs), we are able to predict the fluorescence quenching effect of bringing the E-adenine moiety into close proximity with the indole of tryptophan. (27.6 MPa). The eluate was monitored by absorbance at 254 nm, recorded on a Hewlett-Packard recorder modified as described previously (27). As a criterion of purity we used identical lifetimes determined by both phase shift and modulation (28,29). This technique is very sensitive to the presence of more than a single fluorescent decay, and the presence of EAMP in our preparation would be expected to 3966

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Chloroacetaldehyde-modified dinucleoside phosphates. Dynamic fluorescence quenching and quenching due to intramolecular complexation

Cited by 93 publications

References 41 publications

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Energetics and Specificity of Rat DNA Polymerase β Interactions with Template-primer and Gapped DNA Substrates

Interactions between the Dengue Virus Polymerase NS5 and Stem-Loop A

Dynamic and static quenching of 1,N6-ethenoadenine fluorescence in nicotinamide 1,N6-ethenoadenine dinucleotide and in 1,N6-etheno-9-(3-(indol-3-yl) propyl) adenine.

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