Crystal structure of an RNA·DNA hybrid reveals intermolecular intercalation: Dimer formation by base-pair swapping

Han, Gye Won; Kopka, Mary L.; Langs, D. A.; Sawaya, M.R.; Dickerson, Richard E.

doi:10.1073/pnas.1533326100

Cited by 20 publications

(21 citation statements)

References 53 publications

(53 reference statements)

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“…It is known that anisotropy is related to the transfer angle between donor emission and acceptor absorption as (assuming that the transfer time is much smaller than the time needed for reorientation of dipole moments due to helix twisting) as Simple calculations show that angle u should be close to *45°. In support of our conjecture calculations of the angle between transition dipole moments of the Cy3 and Cy5 fluorophores stacked to the ends of DNA/RNA duplex show that this is close to *500°-508°(sum of Cy3 and Cy5 rotations relative to the end base pairs is equal to 62°, Iqbal et al 2008a, helical twist is equal either 31.3°or 31.9°, Han et al 2003) or, equivalently, to *31°-40°. We note that these data corroborate the assumption about stacking of Cy3 and Cy5 fluorophores to the ends of DNA/ RNA helix which are not impaired by the nature of the linkers used.…”

Section: Discussionsupporting

confidence: 72%

“…where r is the inter-dye distance, N is the number of base pairs, H rise is the rise of DNA/RNA helix (H rise & 3.0 or 2.7 Å (Han et al 2003)) and u is the polar angle between donor and acceptor orientations. L 0 should be considered as the sum of the Cy3 and Cy5 rises (8 Å ; Iqbal et al 2008a) L D and L A can be considered as off-centre distances for donor and acceptor, respectively.…”

Section: Resultsmentioning

confidence: 99%

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Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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Single molecule fluorescent microscopy is a method for the analysis of the dynamics of biological macromolecules by detecting the fluorescence signal produced by fluorophores associated with the macromolecule. Two fluorophores located in a close proximity may result in Förster resonance energy transfer (FRET), which can be detected at the single molecule level and the efficiency of energy transfer calculated. In most cases, the experimentally observed distribution of FRET efficiency exhibits a significant width corresponding to 0.07-0.2 (on a scale of 0-1).Here, we present a general approach describing the analysis of experimental data for a DNA/RNA duplex. We have found that for a 15 bp duplex with Cy3 and Cy5 fluorophores attached to the opposite ends of the helix, the width of the energy transfer distribution is mainly determined by the photon shot noise and the orientation factor, whereas the variation of inter-dye distances plays a minor role.

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Section: Discussionsupporting

confidence: 72%

Section: Resultsmentioning

confidence: 99%

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

2008

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“…The intercalation does not increase base overlap for the base tetrad (Fig. 4), which is different from the base pair swapping in duplex structure (9). The backbone torsion angles of the U1-G2 step of the eight strands are shown in Table 1.…”

Section: Resultsmentioning

confidence: 87%

“…NMR study indicated that cytosine-rich RNA sequences also could form the i-motif in solution, even though they are less stable than their DNA counterparts (8). Recently, intercalation of G⅐C and A⅐T base pairs at the junction of two RNA⅐DNA hybrid duplexes formed a fragment of quadruplex (9).…”

mentioning

confidence: 99%

Base-tetrad swapping results in dimerization of RNA quadruplexes: Implications for formation of thei-motif RNA octaplex

Pan

Shi

Sundaralingam

2006

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

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Nucleic acids adopt different multistranded helical architectures to perform various biological functions. Here, we report a crystal structure of an RNA quadruplex containing ''base-tetrad swapping'' and bulged nucleotide at 2.1-Å resolution. The base-tetrad swapping results in a dimer of quadruplexes with an intercalated octaplex fragment at the 5 end junction. The intercalated base tetrads provide the basic repeat unit for constructing a model of intercalated RNA octaplex. The model we obtained shows fundamentally different characteristics from duplex, triplex, and quadruplex. We also observed two different orientations of bulged uridine residues that are related to the interaction with surroundings. This structural evidence reflects the conformational flexibility of bulged nucleotides in RNA quadruplexes and implies the potential roles of bulged nucleotides as recognition and interaction sites in RNA-protein and RNA-RNA interactions.crystal structure ͉ U(anti)-bulge ͉ G(syn)-tetrad ͉ G(anti)-tetrad

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“…Precisely how the preferred nucleotides facilitate RNase H cleavage is unknown, but it has been proposed that the structures associated with some hybrid sequences might affect cleavage specificity (35). Several features influenced by sequence are the base composition, the trajectory of the helical axis, and the width of the major and minor grooves (35,47,48). Although it is possible that the preferred nucleotides flanking a cleavage site reflect interactions in the DNA strand instead of or in addition to the RNA strand, the co-crystal structure of HIV-1 reverse transcriptase and a PPT-containing RNA/DNA hybrid reveals potential hydrogenbonding contacts between the ϩ1 RNA base and Arg-448, and between the Ϫ2 RNA base and Gln-475 (35) that might contribute to the observed sequence preferences.…”

Section: Volume 281 • Number 4 • January 27 2006mentioning

confidence: 99%

Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H

Schultz

Zhang

Champoux

2006

Journal of Biological Chemistry

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The RNase H activity of reverse transcriptase is essential for retroviral replication. RNA 5-end-directed cleavages represent a form of RNase H activity that is carried out on RNA/DNA hybrids that contain a recessed RNA 5-end. Previously, the distance from the RNA 5-end has been considered the primary determinant for the location of these cleavages. Employing model hybrid substrates and the HIV-1 and Moloney murine leukemia virus reverse transcriptases, we demonstrate that cleavage sites correlate with specific sequences and that the distance from the RNA 5-end determines the extent of cleavage. An alignment of sequences flanking multiple RNA 5-end-directed cleavage sites reveals that both enzymes strongly prefer A or U at the ؉1 position and C or G at the ؊2 position, and additionally for HIV-1, A is disfavored at the ؊4 position. For both enzymes, 5-end-directed cleavages occurred when sites were positioned between the 13th and 20th nucleotides from the RNA 5-end, a distance termed the cleavage window. In examining the importance of accessibility to the RNA 5-end, it was found that the extent of 5-end-directed cleavages observed in substrates containing a free recessed RNA 5-end was most comparable to substrates with a gap of two or three bases between the upstream and downstream RNAs. Together these finding demonstrate that the selection of 5-end-directed cleavage sites by retroviral RNases H results from a combination of nucleotide sequence, permissible distance, and accessibility to the RNA 5-end.In reverse transcription, the single-stranded RNA genome of a retrovirus is transformed into double-stranded DNA by the viral-encoded reverse transcriptase (reviewed in Refs. 1 and 2). This multifunctional enzyme carries out DNA synthesis, strand displacement synthesis, and strand transfer and degrades the RNA portion of RNA/DNA hybrids. The polymerase domain of reverse transcriptase represents the aminoterminal two-thirds of the protein, whereas the RNase H domain comprises the remaining carboxyl-terminal portion. Although the polymerase and RNase H activities are functionally separable, both activities are essential for retroviral replication. The reverse transcriptase of human immunodeficiency virus type 1 (HIV-1) 2 functions as an asymmetric heterodimer composed of p66 and p51 subunits (3), whereas that of Moloney murine leukemia virus (M-MuLV) is a 76-kDa protein that may function as a homodimer or as a monomer (4 -6).RNase H contributes to reverse transcription in three distinct ways (reviewed in Refs. 1, 7, and 8). First, RNase H carries out degradation of the RNA genome both during and after minus-strand DNA synthesis to facilitate plus-strand DNA synthesis and strand transfers. Second, RNase H creates the polypurine tract (PPT) primer from the viral genome. And third, RNase H removes the PPT and tRNA primers used to prime plus-strand and minus-stand DNA synthesis, respectively. Given the vital roles of RNase H in retroviral replication, it is important to understand how RNase H recognizes the RNA/DNA hybri...

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Crystal structure of an RNA·DNA hybrid reveals intermolecular intercalation: Dimer formation by base-pair swapping

Cited by 20 publications

References 53 publications

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Probing complexes with single fluorophores: factors contributing to dispersion of FRET in DNA/RNA duplexes

Base-tetrad swapping results in dimerization of RNA quadruplexes: Implications for formation of thei-motif RNA octaplex

Sequence, Distance, and Accessibility Are Determinants of 5′-End-directed Cleavages by Retroviral RNases H

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