NMR Studies of DNA Support the Role of Pre-Existing Minor Groove Variations in Nucleosome Indirect Readout

We investigated how the intrinsic sequence-dependent properties probed via the phosphate linkages (BI ↔ BII equilibrium) influence the preferred shape of free DNA, and how this affects the nucleosome formation. First, this exploits NMR solution studies of four B-DNA dodecamers that together cover 39 base pairs of the 5′ half of the sequence 601, of special interest for nucleosome formation. The results validate our previous prediction of a systematic, general sequence effect on the intrinsic backbone BII propensities. NMR provides new evidence that the backbone behavior is intimately coupled to the minor groove width. Second, application of the backbone behavior predictions to the full sequence 601 and other relevant sequences demonstrates that alternation of intrinsic low and high BII propensities, coupled to intrinsic narrow and wide minor grooves, largely coincides with the sinusoidal variations of the DNA minor groove width observed in crystallographic structures of the nucleosome. This correspondence is much poorer with low affinity sequences. Overall, the results indicate that nucleosome formation involves an indirect readout process implicating pre-existing DNA minor groove conformations. It also illustrates how the prediction of the intrinsic structural DNA behavior offers a powerful framework to gain explanatory insight on how proteins read DNA

Peptides and oligonucleotides used to prepare and study the epitope.

<p>A: The 29-mer peptide K159 (residues 147–175 in IN of HXB2) was used as immunizing peptide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Sourgen1" target="_blank">[45]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>. To delimit the epitope and analyze the properties we used several other peptides: pep-a4 reproducing the α4-helix sequence; IN636, a C-terminal fragment of K159 that has shown epitope properties in a previous work <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>; IN638, a N-terminal fragment of K159; INH5, a strong inhibitor of IN <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Maroun1" target="_blank">[46]</a>, that includes a loop region (residues 167–171) and the beginning of the α5-helix (residues 172–187); K156, a structural analogue of pep-a4, that is constrained into helix through seven helicogenic substitutions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Zargarian1" target="_blank">[26]</a>; HTH (α4-helix-loop-α5-helix) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016001#pone.0016001-Merad1" target="_blank">[27]</a>; E156, E159 and E148 (structural analogues of K156 in which residues Lys156, Lys159 and Gln148 has been respectively replaced by Glu); K156-E, the elongated K156 peptide ([136–146]-K156); and the peptide control that derives from K156 from six AA→Glu mutations. B: Once folded into hairpin structure around the central trinucleotide <b>TTT</b>, the three LTRoligonucleotides mimic the U5 LTR extremity. LTR17 carries a 7 pb stem corresponding to the most important region for IN binding. LTR34 carries a 17 pb stem, a minimum DNA size for successful reaction of integration <i>in vitro</i>. It represents an unprocessed version of U5 LTR, while LTR32 corresponds to the processed version with its 15 bp and its 5′CA hanging dipeptide. The DNA duplex CRE (cAMP responsive element) is used as a control.</p

Inhibition of binding of MAba4 to IN and K156 by DNA fragments.

<p>A: Histogram representation of competition ELISA results for the competitive binding between DNAs and MAba4 to IN and K156. Wells were coated either with IN and K156 or with the complexes IN-DNA and K156-DNA. IN or K156 where incubated either with DNA or added to the antibody- complex. From left to right: IN, IN-DNA complexes, K156, K156- DNA complexes. Panel values are the mean ± standard deviation (error bars) of three independent experiments. CRE (cAMP Responsive Element) was used as control. B: Histogram representation of simple ELISA results for the binding of DNAs to MAba4. Panel values are the mean ± standard deviation (error bars) of three independent experiments. C: Histogram representation of an ELISA control for the competitive binding of DNAs to IN and K156 realized in presence of a mouse IgA antibody.</p

Effect of MAba4 on the HIV-1 IN activity.

<p>A: Standard concerted integration assays were performed with 1 pmole of IN in presence of increasing amounts of MAba4. The final NaCl concentration was adjusted to 30 mM. MAba4 was added to the mixture at different concentrations and the reaction products were loaded on a 1% agarose gel: 0 (lane 1), 10 (lane 2), 20 (lane 3), 30 (lane 4), 40 (lane 5), 50 (lane 6), 100 (lane 7), 200 lane (8), 600 (lane 9) or 800 ng (lane 10). The position and the structure of the different products obtained after half-site (HSI), full-site (FSI) and donor/donor integration (d/d) are indicated. B: Densitometry of the FSI (full site integration) and FSI+HIS (half site integration) bands of experiments shown in A. The different integration products were quantified using the Image J software. Panel values are the mean ± standard deviation (error bars) of three independent experiments. C: Inhibition assays were performed under different preincubation conditions. MAba4 was either added simultaneously to IN and DNA ([IN+DNA+MAba4]), either after preincubation between IN and DNA ([IN+DNA]+MAba4) or it was preincubated with IN before adding the DNA substrates ([IN+MAba4]+DNA). The different integration products detected on agarose gel were quantified using the Image J software. Panel values are the mean ± standard deviation (error bars) of three independent experiments.</p

Circular dichroism.

<p>Spectra of peptide HTHi at 6 µM (^__, red) and at 25 µM (—, black), and difference spectrum of the complex [HTHi (6 µM)+LTR34 (10 µM)] minus the CD spectrum of LTR34 (10 µM) (……‥, blue). Insert: CD spectrum of HTHi compared to the spectra of its component peptides, α₄ (after Figure 6 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone.0004081-Zargarian1" target="_blank">[43]</a>) and INH5 (after Figure 3b in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone.0004081-Maroun1" target="_blank">[30]</a>). Recall that INH5 comprises both the α₅ helix and the turn linking α₅ to α₄ (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004081#pone-0004081-t001" target="_blank">Table 1a</a>).</p

¹³C transverse relaxation rates (<i>R₁ρ</i>) versus applied spin lock field strength (in Hertz) at 500 MHz for aromatic and anomeric carbons of mini-cTAR DNA. (A) G4 C8; (B) A3 C1′; (C) A21 C8; (D) G16 C1′.

<p>¹³C transverse relaxation rates (<i>R₁ρ</i>) versus applied spin lock field strength (in Hertz) at 500 MHz for aromatic and anomeric carbons of mini-cTAR DNA. (A) G4 C8; (B) A3 C1′; (C) A21 C8; (D) G16 C1′.</p

Gel retardation assays of NC(11–55):mini-cTAR DNA complexes formed <i>in vitro</i>.

<p>(A) Mini-cTAR ³²P-DNAs were incubated in presence of NC(11–55) and analyzed by electrophoresis on a 14% polyacrylamide gel as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038905#s2" target="_blank">Materials and Methods</a>. Lanes 1, controls mini-cTAR dimerization induced by NC(11–55) at a protein to nucleotide molar ratio of 1∶1 (NC(11–55) was removed by phenol/chloroform before gel electrophoresis); lanes 2, heat-denatured mini-cTAR DNAs; lanes 3, controls without protein; lanes 4–7, protein to nucleotide molar ratios were 1∶8, 1∶4, 1∶2 and 1∶1. Monomeric and dimeric forms of free mini-cTAR DNAs are indicated by fm and fd, respectively. CI and CII indicate the NC(11–55):mini-cTAR complexes. (B) Fraction of bound mini-cTAR as a function of the protein:oligonucleotide (expressed in nt) ratio. Each data point represents the mean of three experiments. Symbols: filled circles, mini-cTAR; filled triangles, mini-cTARCT; open triangles, mini-cTARIN2; open circles, mini-cTARIN2CT.</p

Exchange contribution to transverse relaxation versus sequence for aromatic C8 and C6 carbons (A) of mini-cTAR DNA determined from <i>T₁ρ</i> power dependence experiments.

<p>(B) Secondary structure for the mini-cTAR sequence showing the possible transient base-pairs for G4–C22, A5–G20 and C11–G14. The color codes used for the residues are the following: blue (lower stem), orange (internal loop), black (upper stem) and magenta (apical loop).</p

Identification of an “inverted” HTH motif (HTHi) at the catalytic core surface of integrase (PDB ID 1BIU [20]).

<p>a). Crystal structure of the catalytic core domain, associated into a dimer. b). Representation of the HTHi motif, with the loop residues shown by van der Waals spheres. c). The side chain residues involved in intramolecular contacts, shown by sticks and van der Waals spheres. d). The electrostatic potential at the solvent-accessible surface; the Lys-156, Lys-159 and Lys-160 residues are shown by sticks. e). HTHi motif of IN, superimposed onto the “classical” HTH motif of the HMG (highly mobile group) protein LEF-1 (lymphoid enhancer binding factor, PDB ID 2LEF, brown). f). HTHi motif of IN, superimposed onto the HTHi motif of the Signal Recognition Particle (PDB ID 2FFH, green).</p

Imino region of 1D spectra (pH 6.5, in H₂O, 10°C, 60 ms) showing the difference between mini-cTARGC (A) and mini-cTAR (B).

<p>The new resonances observable in the mutant and corresponding to the imino protons of the lower stem are indicated by asteriks.</p