Nucleic acid structure and sequence probing using fluorescent base analogue tCO

Börjesson, Karl; Sandin, Peter; Wilhelmsson, L. Marcus

doi:10.1016/j.bpc.2008.09.021

Cited by 22 publications

(14 citation statements)

References 15 publications

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“…The lifetime of tC o fluorescence depends largely on its local environment. (45–46) We assume that, in a pol-P/T binary complex, the majority of SeqB1 will be in the pol domain, and the majority of SeqB3 will be in the exo domain. If minor-groove HB interactions are important for keeping a P/T in the pol domain, then the local environment of tC o in SeqB2 will resemble that of tC o in SeqB1, otherwise, it will be closer to that of tC o in SeqB3.…”

Section: Results and Disscusionmentioning

confidence: 99%

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

et al. 2012

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Minor groove hydrogen bonding (HB) interactions between DNA polymerases and N3 of purines or O2 of pyrimidines have been proposed to be essential for DNA synthesis from results obtained using various nucleoside analogues lacking the N3 or O2 contacts that interfered with primer-extension. Since there has been no direct structural evidence to support this proposal, we decided to evaluate the contribution of minor groove HB interactions with family B pols. We have used RB69 DNA pol and 3-deaza-2’-deoxyadenosine (3DA), an analog of 2-deoxyadenosine, which has the same HB pattern opposite T but with N3 replaced with a carbon atom. We then determined pre-steady state kinetic parameters for the insertion of dAMP opposite dT using primer/template (P/T) containing 3DA. We also determined three structures of ternary complexes with 3DA at various positions in the duplex DNA substrate. We found that the incorporation efficiency of dAMP opposite dT decreased 102–103 fold even when only one minor groove HB interaction was missing. Our structures show that the HB pattern and base-pair geometry of 3DA/dT is exactly the same as dA/dT, which makes 3DA an optimal analogue for probing minor groove HB interactions between a DNA polymerase and a nucleobase. In addition, our structures provide a rationale for the observed 102–103 fold decrease in nucleotide incorporation. The minor groove HB interactions between the n-2 position of the primer strand and RB69pol fixes the rotomer conformations of the K706 and D621 side chains, as well as the position of metal ion A and its coordinating ligands so that they are in the optinal orientation for DNA synthesis

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Section: Results and Disscusionmentioning

confidence: 99%

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

et al. 2012

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“…It is not surprising that both the replacement of the primary amine of cytosine by a secondary amine and the attachment of an additional aromatic ring alter the electronic character of the N 4 nitrogen. Indeed, we recently reported that a DNA oligonucleotide containing a tC O : A mismatch is significantly more stable than the corresponding duplex with a tC O :T or tC O :C mismatch (51). Moreover, the emission spectrum of the duplex with the tC O :A mismatch exhibited a characteristic fine structure that may point towards firmer base stacking, a change in the polarity of the surroundings, a wobble A + : C base pair resulting from the protonation of adenine at N-1 or hydrogen tautomerisation from the nitrogen at N 4 (pyrimidine numbering) to N-3 of tC O .…”

Section: Resultsmentioning

confidence: 99%

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Sandin

Stengel

Ljungdahl

et al. 2009

Nucleic Acids Research

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Studies of the mechanisms by which DNA polymerases select the correct nucleotide frequently employ fluorescently labeled DNA to monitor conformational rearrangements of the polymerase–DNA complex in response to incoming nucleotides. For this purpose, fluorescent base analogs play an increasingly important role because they interfere less with the DNA–protein interaction than do tethered fluorophores. Here we report the incorporation of the 5′-triphosphates of two exceptionally bright cytosine analogs, 1,3-diaza-2-oxo-phenothiazine (tC) and its oxo-homolog, 1,3-diaza-2-oxo-phenoxazine (tCO), into DNA by the Klenow fragment. Both nucleotide analogs are polymerized with slightly higher efficiency opposite guanine than cytosine triphosphate and are shown to bind with nanomolar affinity to the DNA polymerase active site, according to fluorescence anisotropy measurements. Using this method, we perform competitive binding experiments and show that they can be used to determine the dissociation constant of any given natural or unnatural nucleotide. The results demonstrate that the active site of the Klenow fragment is flexible enough to tolerate base pairs that are size-expanded in the major groove. In addition, the possibility to enzymatically polymerize a fluorescent nucleotide with high efficiency complements the tool box of biophysical probes available to study DNA replication.

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“…In a 10 base pair duplex, changing the base pair partner with tCo gave T M s of tCo-G 46 °C, tCo-A 34 °C, tCo-T 24 °C and tCo -C 20°C. Thus, the tCo-A mismatch is significantly more stable than the other mismatches (41). The fluorescence emission spectrum also mirrors the special character of the tCo-A base pair.…”

Section: Discussionmentioning

confidence: 99%

“…The fluorescence emission spectrum also mirrors the special character of the tCo-A base pair. It exhibits a fine structure that could be indicative of a stabilized imino tautomer, besides other structural rearrangements (41). Interestingly, the thermodynamic mismatch stabilities correlate with the order of mismatch discrimination by pol α and KF.…”

Section: Discussionmentioning

confidence: 99%

Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase α and Klenow Fragment

et al. 2009

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We studied the incorporation of the fluorescent cytidine analogues 1, 3-diaza-2-oxo-phenothiazine (tC) and 1, 3-diaza-2-oxo-phenoxazine (tCo) by human DNA polymerase α and Klenow fragment of DNA polymerase I (E. coli). These tricyclic nucleobases possess the regular hydrogen bonding interface of cytosine but are significantly size expanded toward the major groove. Despite the size alteration both DNA polymerases insert dtCTP and dtCoTP with remarkable catalytic efficiency. Polymerization opposite guanine is comparable to the insertion of dCTP, while the insertion opposite adenine is only ∼4-11 times less efficient than the formation of a T-A base pair. Both enzymes readily extend the formed tC(o)-G and tC(o)-A base pairs, and can incorporate at least 4 consecutive nucleotide analogues. Consistent with these results, both DNA polymerases efficiently polymerize dGTP and dATP when tC and tCo are in the template strand. KF inserts dGTP with a 4-to 9-fold higher probability than dATP, while pol α favors dGTP over dATP by a factor of 30-65. Overall, the properties of tC(o) as templating base and as incoming nucleotide are surprisingly symmetrical and may be universal for A and B family DNA polymerases. This finding suggests that the aptitude for ambivalent base pairing is a consequence of the electronic properties of tC(o).

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Nucleic acid structure and sequence probing using fluorescent base analogue tCO

Cited by 22 publications

References 15 publications

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

Probing Minor Groove Hydrogen Bonding Interactions between RB69 DNA Polymerase and DNA

Highly efficient incorporation of the fluorescent nucleotide analogs tC and tCO by Klenow fragment

Ambivalent Incorporation of the Fluorescent Cytosine Analogues tC and tCo by Human DNA Polymerase α and Klenow Fragment

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