Optical tweezers experiments resolve distinct modes of DNA-protein binding.

Abstract:

:Optical tweezers are ideally suited to perform force microscopy experiments that isolate a single biomolecule, which then provides multiple binding sites for ligands. The captured complex may be subjected to a spectrum of forces, inhibiting or facilitating ligand activity. In the following experiments, we utilize optical tweezers to characterize and quantify DNA binding of various ligands. High mobility group type B (HMGB) proteins, which bind to double-stranded DNA, are shown to serve the dual purpose of stabilizing and enhancing the flexibility of double stranded DNA. Unusual intercalating ligands are observed to thread into and lengthen the double-stranded structure. Proteins binding to both double- and single-stranded DNA, such as the alpha polymerase subunit of E. coli Pol III, are characterized, and the subdomains containing the distinct sites responsible for binding are isolated. Finally, DNA binding of bacteriophage T4 and T7 single-stranded DNA (ssDNA) binding proteins is measured for a range of salt concentrations, illustrating a binding model for proteins that slide along double-stranded DNA, ultimately binding tightly to ssDNA. These recently developed methods quantify both the binding activity of the ligand as well as the mode of binding.

journal_name

Biopolymers

journal_title

Biopolymers

authors

McCauley MJ,Williams MC

doi

10.1002/bip.21123

subject

Has Abstract

pub_date

2009-04-01 00:00:00

pages

265-82

issue

4

eissn

0006-3525

issn

1097-0282

journal_volume

91

pub_type

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