Transient HMGB protein interactions with B-DNA duplexes and complexes

Abstract

HMGB proteins are abundant, non-histone proteins in eukaryotic chromatin. HMGB proteins contain one or two conserved "HMG boxes" and can be sequence specific or nonspecific in their DNA binding. HMGB proteins cause strong DNA bending and bind preferentially to deformed DNAs. We wish to understand how HMGB proteins increase the apparent flexibility of non-distorted B-form DNA. We test the hypothesis that HMGB proteins bind transiently, creating an ensemble of distorted DNAs with rapidly-interconverting conformations. We show that binding of B-form DNA by HMGB proteins is both weak and transient under conditions where DNA cyclization is strongly enhanced. We also detect novel complexes in which HMGB proteins simultaneously bind more than one DNA duplex.Double-stranded DNA is among the least flexible biopolymers. The local stiffness of DNA is reflected in its persistence length, P, (~150 bp) [1]. The global flexibility of naked DNA in solution is insufficient to allow the degree of compaction required for packaging within cells, or for deformed structures involved in DNA replication, recombination, and transcriptional control. While some controversy exists concerning the accuracy of predictive models for DNA flexibility over short lengths [2;3;4], it is clear that short lengths of DNA require proteins to induce strongly bent and looped conformations.The apparent physical properties of DNA may differ in vitro and in vivo [5;6]. Activation over short distances in assays of eukaryotic transcription activation in vitro raised the possibility of apparent DNA flexibility enhancement by nuclear factors [7]. Indeed, heat-resistant HMGB proteins in nuclear extracts were found to dramatically enhance the cyclization of short DNA restriction fragments by DNA ligase [8].Eukaryotic HMGB proteins are abundant small non-histone chromosomal proteins [9;10;11; 12] that share one or two copies of an ~80-amino acid HMG box domain. Fig. 1A shows the amino acid alignment of HMG box domains, illustrating the partial conservation of intercalating residues [13]. As shown in Fig. 1B, this domain specifies three α-helices (red) whose L-shaped fold engages one face of DNA while delivering one or two intercalating residues into the minor groove [14;15;16]. Biophysical studies have provided some insights into the thermodynamic basis of DNA binding [17;18;19;20]. HMGB proteins to induce DNA kinking or bending of 22;23;24;25], and bind preferentially to distorted DNA structures [16;21;26]. HMGB proteins may stabilize DNA bending either by *To whom correspondence should be addressed at maher@mayo.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all l...

DNA stretching as a probe for nucleic acid interactions

“…Quantifying this change with increasing protein concentrations reveals the binding affinity, and can also provide insight into the specific binding mode. In the case of HMG, average induced bending angles were determined from changes in the persistence length [12], and were in good agreement with other results, including direct visualization methods of AFM [13], as well as cyclization measurements that clearly demonstrate an increase in apparent DNA flexibility induced by these proteins [14]. Distributions of the bending angles seen in AFM experiments further reveal that this change in the persistence length is driven by a combination of discrete bends (static kinks), and increased flexibility at the binding site (flexible hinges) [13].…”

DNA-Segment-Facilitated Dissociation of Fis and NHP6A from DNA Detected via Single-Molecule Mechanical Response

“…In this paper we have studied similar “facilitated dissociation” effects driven by solution phase DNA segments; the presence of DNA segments accelerates the release of pre-bound Fis from a DNA molecule in the manner of “direct transfer” reactions studied for other proteins exchanging between nucleic acid molecules 10; 12; 13; 14; 15; 16; 17; 18; 19; 20; 21; 22; 23; 24; 25; 26; 27; 28 . We have done most of our experiments using the DNA-compacting effect of Fis as a readout of binding (Fig.…”

“…Double-helix to double-helix “direct transfer” processes have been observed for a variety of DNA-binding ligands and proteins, including ethidium bromide 13; 14 , bacterial transcription factors CAP and Lac repressor 15; 16 , restriction enzymes 17 , eukaryote transcription factors 18; 19; 20 , HMGB proteins 21 , and histones 22; 23 . Direct transfer kinetics also have been observed for transfer of proteins between ssDNA segments, notably for RecA 24 and SSB 25; 26; 27 .…”

The HMGB1 C-Terminal Tail Regulates DNA Bending

“…switching between a bent FRET state and an unbent FRET state on a single molecule of DNA over time was seldom observed). Some studies have shown that DNA binding by HMGB1 is dynamic in vitro [30,31] and also in cells [32], while other studies measured slower rates of dissociation [33]. A recent single molecule kinetic study using optical tweezers proposed that microscopic and macroscopic dissociation constants control interactions between HMGB proteins and DNA [34].…”