Experimental maps of DNA structure at nucleotide resolution distinguish intrinsic from protein-induced DNA deformations

Abstract Recognition of DNA by proteins depends on DNA sequence and structure. Often unanswered is whether the structure of naked DNA persists in a protein–DNA complex, or whether protein binding changes DNA shape. While X-ray structures of protein–DNA complexes are numerous, the structure of naked cognate DNA is seldom available experimentally. We present here an experimental and computational analysis pipeline that uses hydroxyl radical cleavage to map, at single-nucleotide resolution, DNA minor groove width, a recognition feature widely exploited by proteins. For 11 protein–DNA complexes, we compared experimental maps of naked DNA minor groove width with minor groove width measured from X-ray co-crystal structures. Seven sites had similar minor groove widths as naked DNA and when bound to protein. For four sites, part of the DNA in the complex had the same structure as naked DNA, and part changed structure upon protein binding. We compared the experimental map with minor groove patterns of DNA predicted by two computational approaches, DNAshape and ORChID2, and found good but not perfect concordance with both. This experimental approach will be useful in mapping structures of DNA sequences for which high-resolution structural data are unavailable. This approach allows probing of protein family-dependent readout mechanisms.

plates containing 100 µg/mL ampicillin sodium salt (Sigma Aldrich). Plates were incubated overnight at 37°C. Individual colonies were selected from a plate and used to separately inoculate 5-8 mL starter cultures containing LB broth and 100 µg/mL ampicillin. Cultures were grown overnight at 37°C with continuous shaking. Plasmid DNA was isolated from the cell culture using the QIAprep Miniprep kit (Qiagen) following the manufacturer's recommended protocol. Plasmid DNA was eluted from the spin column with 50 µL Buffer EB (10 mM Tris·Cl, pH 8.5), with typical recoveries of ~150 ng/µL DNA.
PCR amplification and purification of 5' end-labeled DNA PCR amplification of the insert sequence from whole plasmid DNA was performed in a 96-well plate in a reaction volume of 40 µL per well. The PCR reaction mixture was prepared in a single 1.5 mL microcentrifuge tube, on ice, in a total volume of 320 µL, and then aliquoted into 8 wells of a 96-well plate. Each reaction contained 1X ThermoPol buffer (New England Biolabs), 10% DMSO (vol/vol), 3 mM MgSO 4 , 200 µM dNTP mix, 0.5 µM P3F and P3R primers, 200 ng DNA template, and 0.3-0.6 U Vent Polymerase (New England Biolabs). PCR thermal-cycling was performed using an initial denaturation step at 90°C for 3 min 45 s, followed by 34 cycles of annealing at 52°C for 40 s and extension at 72°C for 30 s. A final extension step was carried out at 72°C for 5 min before cooling to 4°C for storage. To synthesize a fluorescently labeled PCR product, one of the two primers in the reaction mixture contained Cy5 dye covalently attached at the 5' end, while the other primer was unlabeled.
Post-PCR reaction cleanup was performed using a Biomek 3000 Automated Workstation (Beckman Coulter) equipped with a multi-channel pipet tool and a gripper for 96-well plates. Purification of the PCR product was fully automated and achieved using Agencourt AMPure XP magnetic beads (Beckman Coulter). The Biomek 3000 was programmed to follow the Agencourt-recommended protocol, which consisted of adding 72 µL of resuspended bead solution directly to the PCR mixture, mixing 10 times with a pipet tip, and incubating at room temperature for 5 min. The sample plate was then moved onto a magnet designed to accommodate 96-well plates and allowed to sit for 3 min while the beads were pulled to the inner walls of the wells. The supernatant was discarded and the beads were rinsed 2-3 times with freshly prepared 70% ethanol. After air-drying for 10 min, the plate was transferred off the magnet and the beads were resuspended in 40 µL TE buffer (pH 8.0) to elute the DNA. After 3 min incubation, the plate was transferred back to the magnet, the beads were separated, and the supernatant containing the purified DNA was transferred to a fresh 96-well plate. Absorbance measurements at 260 nm were acquired using a Nanodrop system to quantify the total amount of DNA recovered, typically 20-30 ng/µL. Supplementary Table S1. Sequences of DNA binding sites in protein-DNA co-crystal structures.

Protein
Binding Site Sequence PDB ID

SUPPLEMENTARY FIGURES
Supplementary Figure S1. Loess smoothing of the experimental ORChID2 pattern. Grey, raw experimental ORChID2 data; black, ORChID2 data smoothed by the loess.smooth function in R. 300 data points (nucleotides) were smoothed, using parameters span=0.015 and evaluation=300.
Supplementary Figure S2. Comparison of the experimental ORChID2 pattern with the minor groove width pattern determined by X-ray crystallography for the Drew-Dickerson dodecamer sequence. The experimental ORChID2 pattern (red) was taken from the dataset obtained for the 399-bp DNA molecule (see Fig. 1). The minor groove width pattern (blue) was determined by averaging and symmetrizing minor groove width measurements made on eight Xray crystal structures of the Drew-Dickerson dodecamer. The Spearman's rank correlation coefficient r for comparison of these two patterns was 0.97. Grey, computed ORChID2 data; black, experimental ORChID2 data. Both patterns were smoothed by the loess.smooth function in R. 300 data points (nucleotides) were smoothed, using parameters span=0.015 and evaluation=300.

Supplementary
Supplementary Figure S6. The minor groove width patterns of the left and right segments of the Oct-1 (PORE) site did not change upon protein binding. Red filled circles, expORChID2 values; blue filled triangles, minor groove width measured from the protein-DNA complex; teal filled squares, minor groove width predicted by DNAshape for naked DNA. Arrows, locations of arginine residues bound to the minor groove in the protein-DNA complex,