title: Single-molecule kinetics reveal microscopic mechanism by which High-Mobility Group B proteins alter DNA flexibility.
abstract: 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.
authors.affiliation: Department of Physics, Northeastern University, Boston, MA 02115, USA.
authors.forename: Micah J
authors.initials: MJ
authors.lastname: McCauley
authors.forename: Emily M
authors.initials: EM
authors.lastname: Rueter
authors.forename: Ioulia
authors.initials: I
authors.lastname: Rouzina
authors.forename: L James
authors.initials: LJ
authors.lastname: Maher
authors.forename: Mark C
authors.initials: MC
authors.lastname: Williams
mesh_terms: D000595:Amino Acid Sequence; D020029:Base Pairing; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D059371:DNA, B-Form / Q000737:chemistry; D004548:Elasticity; D024742:HMG-Box Domains; D024002:HMGB Proteins / Q000737:chemistry* / Q000378:metabolism; D024261:HMGB2 Protein / Q000737:chemistry / Q000378:metabolism; D024001:HMGN Proteins / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
chemical_list: D059371:DNA, B-Form; D024002:HMGB Proteins; D024261:HMGB2 Protein; D024001:HMGN Proteins; C086471:NHP6A protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA