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

Micah J. McCauley; Emily M. Rueter; Ioulia Rouzina; L. James Maher; Mark C. Williams

doi:10.1093/nar/gks1031

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

Micah J. McCauley, Emily M. Rueter, Ioulia Rouzina, L. James Maher, Mark C. Williams

Biochemistry and Molecular Biology

Research output: Contribution to journal › Article › peer-review

30 Scopus citations

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.

Original language	English (US)
Pages (from-to)	167-181
Number of pages	15
Journal	Nucleic acids research
Volume	41
Issue number	1
DOIs	https://doi.org/10.1093/nar/gks1031
State	Published - Jan 2013

ASJC Scopus subject areas

Genetics

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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.",

author = "McCauley, {Micah J.} and Rueter, {Emily M.} and Ioulia Rouzina and Maher, {L. James} and Williams, {Mark C.}",

note = "Funding Information: National Institutes of Health [2R01GM72462 to M.C.W., 2R01GM075965 to L.J.M.]; Mayo Foundation. Funding for open access charge: NIH [GM075965].",

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doi = "10.1093/nar/gks1031",

language = "English (US)",

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AU - Rouzina, Ioulia

AU - Maher, L. James

AU - Williams, Mark C.

N1 - Funding Information: National Institutes of Health [2R01GM72462 to M.C.W., 2R01GM075965 to L.J.M.]; Mayo Foundation. Funding for open access charge: NIH [GM075965].

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N2 - 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.

AB - 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.

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

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