Single-molecule FRET analysis of DNA binding and bending by yeast HMGB protein Nhp6A.

High-mobility group B (HMGB) proteins bind duplex DNA without sequence specificity, facilitating the formation of compact nucleoprotein structures by increasing the apparent flexibility of DNA through the introduction of DNA kinks. It has remained unclear whether HMGB binding and DNA kinking are simultaneous and whether the induced kink is rigid (static) or flexible. The detailed molecular mechanism of HMGB-induced DNA 'softening' is explored here by single-molecule fluorescence resonance energy transfer studies of single yeast Nhp6A (yNhp6A) proteins binding to short DNA duplexes. We show that the local effect of yNhp6A protein binding to DNA is consistent with formation of a single static kink that is short lived (lifetimes of a few seconds) under physiological buffer conditions. Within the time resolution of our experiments, this static kink occurs at the instant the protein binds to the DNA, and the DNA straightens at the instant the protein dissociates from the DNA. Our observations support a model in which HMGB proteins soften DNA through random dynamic binding and dissociation, accompanied by DNA kinking and straightening, respectively.

Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility.

Eukaryotic High-Mobility Group B (HMGB) proteins alter DNA elasticity while facilitating transcription, replication and DNA repair. We developed a new single-molecule method to probe non-specific DNA interactions for two HMGB homologs: the human HMGB2 box A domain and yeast Nhp6Ap, along with chimeric mutants replacing neutral N-terminal residues of the HMGB2 protein with cationic sequences from Nhp6Ap. Surprisingly, HMGB proteins constrain DNA winding, and this torsional constraint is released over short timescales. These measurements reveal the microscopic dissociation rates of HMGB from DNA. Separate microscopic and macroscopic (or local and non-local) unbinding rates have been previously proposed, but never independently observed. Microscopic dissociation rates for the chimeric mutants (~10 s(-1)) are higher than those observed for wild-type proteins (~0.1-1.0 s(-1)), reflecting their reduced ability to bend DNA through short-range interactions, despite their increased DNA-binding affinity. Therefore, transient local HMGB-DNA contacts dominate the DNA-bending mechanism used by these important architectural proteins to increase DNA flexibility.

Quantitative methods for measuring DNA flexibility in vitro and in vivo.

The double-helical DNA biopolymer is particularly resistant to bending and twisting deformations. This property has important implications for DNA folding in vitro and for the packaging and function of DNA in living cells. Among the outstanding questions in the field of DNA biophysics are the underlying origin of DNA stiffness and the mechanisms by which DNA stiffness is overcome within cells. Exploring these questions requires experimental methods to quantitatively measure DNA bending and twisting stiffness both in vitro and in vivo. Here, we discuss two classical approaches: T4 DNA ligase-mediated DNA cyclization kinetics and lac repressor-mediated DNA looping in Escherichia coli. We review the theoretical basis for these techniques and how each can be applied to quantitate biophysical parameters that describe the DNA polymer. We then show how we have modified these methods and applied them to quantitate how apparent DNA physical properties are altered in vitro and in vivo by sequence-nonspecific architectural DNA-binding proteins such as the E. coli HU protein and eukaryotic HMGB proteins.

Understanding apparent DNA flexibility enhancement by HU and HMGB architectural proteins.

Understanding and predicting the mechanical properties of protein/DNA complexes are challenging problems in biophysics. Certain architectural proteins bind DNA without sequence specificity and strongly distort the double helix. These proteins rapidly bind and unbind, seemingly enhancing the flexibility of DNA as measured by cyclization kinetics. The ability of architectural proteins to overcome DNA stiffness has important biological consequences, but the detailed mechanism of apparent DNA flexibility enhancement by these proteins has not been clear. Here, we apply a novel Monte Carlo approach that incorporates the precise effects of protein on DNA structure to interpret new experimental data for the bacterial histone-like HU protein and two eukaryotic high-mobility group class B (HMGB) proteins binding to ∼200-bp DNA molecules. These data (experimental measurement of protein-induced increase in DNA cyclization) are compared with simulated cyclization propensities to deduce the global structure and binding characteristics of the closed protein/DNA assemblies. The simulations account for all observed (chain length and concentration dependent) effects of protein on DNA behavior, including how the experimental cyclization maxima, observed at DNA lengths that are not an integral helical repeat, reflect the deformation of DNA by the architectural proteins and how random DNA binding by different proteins enhances DNA cyclization to different levels. This combination of experiment and simulation provides a powerful new approach to resolve a long-standing problem in the biophysics of protein/DNA interactions.