Analysis of scanning force microscopy images of protein-induced DNA bending using simulations

Bending of DNA is a feature essential to the function of many DNA-binding proteins. Bending angles can be estimated with a variety of techniques, but most directly from images obtained using scanning force microscopy (SFM). Direct measurement of the bending angle using a tangent method often produces angles that deviate significantly from values obtained using other techniques. Here, we describe the application of SFM in combination with simulations of DNA as a means to estimate protein-induced bending angles in a reliable and unbiased fashion. In this manner, we were able to obtain accurate estimates for the bending angles induced by nuclear factor I, octamer-binding transcription factor 1, the human XPC-Rad23B complex

NFI and Oct-1 bend the Ad5 origin in the same direction leading to optimal DNA replication

Two cellular transcription factors, nuclear factor I (NFI) and octamer binding protein (Oct-1), bind simultaneously to their recognition sequences in the Ad5 origin of replication thereby enhancing initiation. Using scanning force microscopy we have previously shown that NFI induces a 60° bend in the origin DNA. Here we demonstrate that Oct-1 induces a 42° bend in the origin DNA. Simultaneous binding of NFI and Oct-1 induces an 82° collective bend suggesting that both bends are oriented towards each other. In functional replication assays we further demonstrate that this extensive DNA bending leads to a synergistic enhancement of DNA replication. We propose that collective DNA bending induced by NFI and Oct-1 facilitates the optimal assembly of the preinitiation complex and plays an important role in the stimulatory mechanism of NFI and Oct-1 in replication

Bending of Adenovirus Origin DNA by Nuclear Factor I as Shown by Scanning Force Microscopy Is Required for Optimal DNA Replication

Nuclear factor I (NFI) is a transcription factor that binds to the adenovirus type 5 (Ad5) origin of replication and recruits the adenovirus DNA polymerase, thereby stimulating initiation of DNA replication in vitro. Using scanning force microscopy, we demonstrate that NFI induces a 60° bend upon binding to the origin. The A/T-rich region preceding the core recognition sequence of NFI influences the DNA bend angle, since substitution of A/T base pairs by G/C base pairs severely decreases bending. Mutations in the A/T-rich region do not affect binding of NFI to DNA. However, mutations that reduce the protein-induced bend lead to a loss of NFI-stimulated replication, indicating that DNA bending is functionally important. In contrast, basal initiation or DNA binding of the polymerase is not impaired by these origin mutations. We conclude that binding of NFI to the Ad5 origin causes structural changes in DNA that are essential for the stimulatory function of NFI in replication. We propose that NFI-induced origin bending facilitates the assembly of a functional initiation complex

() Typical IHF–DNA complexes imaged as described in Materials and Methods

<p><b>Copyright information:</b></p><p>Taken from "Analysis of scanning force microscopy images of protein-induced DNA bending using simulations"</p><p>Nucleic Acids Research 2005;33(7):e68-e68.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083423.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> The protein-induced bend is indicated by the arrows. Owing to its size, the IHF protein cannot be unambiguously identified in the images, demonstrating the need for an analytical approach using other than visual characteristics. The scale bar is 50 nm. Gray scale represents height ranging from 0 nm (dark) to 2 nm (bright). () Distributions of EED values normalized by contour lengths of IHF–DNA complexes (top) and bare DNA molecules (bottom), demonstrating the effect of DNA bending. () Histogram of bending angles estimated using tangents from IHF–DNA complexes. The bimodal distribution shows that not all DNA molecules have IHF bound; by fitting to a double Gaussian distribution, we estimate that ∼50% of the imaged molecules have IHF bound

(–) Histograms of experimentally obtained values for EED normalized by contour lengths of bare DNA molecules and protein–DNA complexes and the corresponding fits

<p><b>Copyright information:</b></p><p>Taken from "Analysis of scanning force microscopy images of protein-induced DNA bending using simulations"</p><p>Nucleic Acids Research 2005;33(7):e68-e68.</p><p>Published online 20 Apr 2005</p><p>PMCID:PMC1083423.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> (–) χ profiles for the data sets (solid lines with squares). The intersections with the dashed line indicate the uncertainty in the angle determination ()