Fluorescence Properties of Pteridine Nucleoside Analogs as Monomers and Incorporated into Oligonucleotides

Hawkins, Mary E.; Pfleiderer, Wolfgang; Balis, Frank M.; Porter, Denise; Knutson, Jay R.

doi:10.1006/abio.1996.9879

Cited by 155 publications

(230 citation statements)

References 13 publications

(18 reference statements)

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“…Incorporation of the 3-methyl-8-(2-deoxy-␤-D-ribofuranosyl) isoxanthopterin (3MI) probe into ODN and fluorescence properties of the PTER1 ODN (Fig. 1G) have been described (19,20). Double-stranded ODNs were obtained by mixing equimolar amounts of complementary strands in a 20 mM Tris⅐HCl buffer (pH 7.2) containing 100 mM NaCl.…”

Section: Methodsmentioning

confidence: 99%

“…The experiments described above with fluorescein-labeled ODNs were repeated with the 21-mer, basemodified ODN, PTER1 (Fig. 1G), in which the guanosine at position 19 (from the 3Ј end) was replaced by the fluorescent guanosine analog, 3MI (19,20). This substitution had no detectable effect on either 3Ј processing or integration reaction (not shown).…”

Section: Determination Of the Of The In-dna Complex Using A Base-modimentioning

confidence: 97%

See 1 more Smart Citation

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Deprez

Tauc

Leh

et al. 2001

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

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Self-assembly of HIV-1 integrase (IN) in solution has been studied previously by time-resolved fluorescence, using tryptophan anisotropy decay. This approach provides information on the size of macromolecules via the determination of rotational correlation times ( ). We have shown that, at submicromolar concentration, IN is characterized by a long rotational correlation time ( 20°C ‫؍‬ 90 -100 ns) corresponding to a high-order oligomeric form, likely a tetramer. In the present work, we investigated the self-assembly properties of the DNA-bound IN by using three independent fluorophores. Under enzymatic assay conditions (10 ؊7 M IN, 2 ؋ 10 ؊8 M DNA), using either fluorescein-labeled or fluorescent guanosine analog-containing oligonucleotides that mimic a viral end long terminal repeat sequence, we found that the DNA- I ntegration of a DNA copy of the HIV-1 genome into the host genome is a critical step of the retrovirus life cycle. Integration is a multistep process including 3Ј processing, strand transfer, and DNA repair. Integrase (IN) is responsible for 3Ј processing and strand transfer and is sufficient to catalyze these two reactions in vitro, using short-length oligonucleotides (ODNs) that mimic the viral end long terminal repeat sequences. The repair step most likely is performed by a host system. IN is a member of the superfamily of polynucleotidyl transferases, and its catalytic core domain contains a triad of acidic residues that constitute the so-called D,D-35-E motif. This motif is strictly required for catalysis (1, 2) and is involved in the coordination of the metal cation cofactor (3, 4). Catalysis can be performed efficiently in vitro by using either Mn 2ϩ or Mg 2ϩ , but several investigations suggest that these two divalent cations have different effects on the reaction specificity. Specific protein-DNA contacts occur preferentially in the presence of Mg 2ϩ (5), and more nonspecific cleavage is detected in the presence of Mn 2ϩ (6). The transfer pattern on the target DNA also depends on the nature of the cation (7). Consequently, the efficiency of inhibitors can be different by using either Mg 2ϩ or Mn 2ϩ in assays (8). In each case, the Mg 2ϩ -dependent activity may be more reliable because it is more physiologically relevant.Specific recognition between IN and the substrate viral DNA is a prerequisite for the cleavage of two nucleotides at the 3Ј end of the viral DNA. The catalytic core domain establishes specific contacts with the viral DNA, and it has been determined that the terminal 13-base region of the long terminal repeat plays a significant role for Mg 2ϩ -dependent processing in terms of specificity (5, 9). The C-terminal domain also mediates viral DNA-IN interactions, but in a nonspecific fashion, and more likely contributes to complex stabilization (10-12). Furthermore, it is strongly believed that self-association of IN plays a key role in governing the enzyme function (13-16). Recently, we have shown that the capability of IN to use Mg 2ϩ is related to the protein self-association ...

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Section: Methodsmentioning

confidence: 99%

Section: Determination Of the Of The In-dna Complex Using A Base-modimentioning

confidence: 97%

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Deprez

Tauc

Leh

et al. 2001

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

Self Cite

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“…We have used the available high resolution structures of UP1 to incorporate the fluorescent guanosine analog 6-methyl-8-(2-deoxy-␤-ribofuranosyl)isoxanthopteridine (6-MI) into the oligonucleotide d(TTAGGG) 2 as a probe for studying UP1 interactions to hTR DNA (33,34). Previous fluorescence studies using either the quenching of intrinsic tryptophan fluorescence or the fluorescent nucleotide riboethanoadenylic acid have shown that UP1 binds promiscuously to single-stranded nucleic acids as part of its in vivo role in RNA processing and transport (22,23,27).…”

mentioning

confidence: 99%

Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Myers

Moore

Shamoo

2003

Journal of Biological Chemistry

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Heterogeneous ribonucleoprotein A1 (hnRNP A1) is an abundant nuclear protein that participates in RNA processing, alternative splicing, and chromosome maintenance. hnRNP A1 can be proteolyzed to unwinding protein (UP1), a 22.1-kDa protein that retains a high affinity for purine-rich single-stranded nucleic acids, including the human telomeric repeat (hTR) d(T-TAGGG) n . Using the structure of UP1 bound to hTR as a guide, we have incorporated the fluorescent guanine analog 6-MI at one of two positions within the DNA to facilitate binding studies. One is where 6-MI remains stacked with an adjacent purine, and another is where it becomes fully unstacked upon UP1 binding. The structures of both modified oligonucleotides complexed to UP1 were determined by x-ray crystallography to validate the efficacy of our design, and 6-MI has proven to be an excellent reporter molecule for single-stranded nucleic acid interactions in positions where there is a change in stacking environment upon complex formation. We have shown that UP1 affinity for d(TTAGGG) 2 is ϳ5 nM at 100 mM NaCl, pH 6.0, and our binding studies with d(TTAGG(6-MI)TTAGGG) show that binding is only modestly sensitive to salt and pH. UP1 also has a potent G-tetrad destabilizing activity that reduces the T m of the hTR sequence d(TAGGGT) 4 from 67.0°C to 36.1°C at physiological conditions (150 mM KCl, pH 7.0). Consistent with the structures determined by x-ray crystallography, UP1 is able to bind the hTR sequence in solution as a dimer and supports a model for hnRNP A1 binding to nucleic acids in arrays that may make a contiguous set of anti-parallel single-stranded nucleic acid binding clefts. These data suggest that seemingly disparate roles for hnRNP A1 in alternative splice site selection, RNA processing, RNA transport, and chromosome maintenance reflect its ability to bind a purine-rich consensus sequence (nYAGGn) and destabilize potentially deleterious G-tetrad structures.

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“…6 Table 1 The experiments described above used nonfluorescent oligonucleotides, as we initially assumed that the ATP hydrolysis activity of the 6MI-labeled oligonucleotides would be reasonably approximated by the equivalent nonfluorescent oligonucleotides since the 6MI fluorophore does not perturb the DNA structure. 55 In fact, ATPase experiments performed with oligonucleotide 30-6MI confirmed this assumption (data not shown). In contrast to the 6MI-labeled and non-fluorescent oligonucleotides, the dissociation constant determined using the 5′-fluorescein-labeled 30mer shows that the extrinsic label has a significant effect on the activity of RecA (Table 1).…”

Section: Atp Hydrolysis Activity Using Short Oligonucleotidesmentioning

confidence: 76%

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

Singleton¹,

Roca²,

Lee³

et al. 2007

Tetrahedron

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The RecA protein of Escherichia coli plays a crucial roles in DNA recombination and repair, as well as various aspects of bacterial pathogenicity. The formation of a RecA-ATP-ssDNA complex initiates all RecA activities and yet a complete structural and mechanistic description of this filament has remained elusive. An analysis of RecA-DNA interactions was performed using fluorescently labeled oligonucleotides. A direct comparison was made between fluorescein and several fluorescent nucleosides. The fluorescent guanine analog 6-methylisoxanthopterin (6MI) demonstrated significant advantages over the other fluorophores and represents an important new tool for characterizing RecA-DNA interactions.

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Fluorescence Properties of Pteridine Nucleoside Analogs as Monomers and Incorporated into Oligonucleotides

Cited by 155 publications

References 13 publications

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

DNA binding induces dissociation of the multimeric form of HIV-1 integrase: A time-resolved fluorescence anisotropy study

Structure-based Incorporation of 6-Methyl-8-(2-deoxy-β-ribofuranosyl)isoxanthopteridine into the Human Telomeric Repeat DNA as a Probe for UP1 Binding and Destabilization of G-tetrad Structures

Probing the structure of RecA–DNA filaments. Advantages of a fluorescent guanine analog

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