Single Molecule Force Spectroscopy of Salt-dependent Bacteriophage T7 Gene 2.5 Protein Binding to Single-stranded DNA

Leila Shokri,Charles C Richardson,Ioulia Rouzina,Boriana Marintcheva,Mark C Williams

doi:10.1074/jbc.m608460200

Leila Shokri, Charles C Richardson + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m608460200

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Abstract

The gene 2.5 protein (gp2.5) encoded by bacteriophage T7 binds preferentially to single-stranded DNA. This property is essential for its role in DNA replication and recombination in the phage-infected cell. gp2.5 lowers the phage lambda DNA melting force as measured by single molecule force spectroscopy. T7 gp2.5-Delta26C, lacking 26 acidic C-terminal residues, also reduces the melting force but at considerably lower concentrations. The equilibrium binding constants of these proteins to single-stranded DNA (ssDNA) as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 +/- 0.6) x 10(5) m(-1) in a 50 mm Na(+) solution, whereas the truncated protein binds to ssDNA with a much higher affinity of (7.8 +/- 0.9) x 10(7) m(-1) under the same solution conditions. T7 gp2.5-Delta26C binding to single-stranded DNA also exhibits a stronger salt dependence than the full-length protein. The data are consistent with a model in which a dimeric gp2.5 must dissociate prior to binding to ssDNA, a dissociation that consists of a weak non-electrostatic and a strong electrostatic component.

Highlights

Optical tweezers have been used extensively for studying the biomechanical properties of single DNA molecules by stretching the molecules and measuring the required force for a given extension under various conditions [1,2,3,4,5,6]
The equilibrium binding constants of these proteins to singlestranded DNA as a function of salt concentration have been determined, and we found for example that gp2.5 binds with an affinity of (3.5 ؎ 0.6) ؋ 105 M؊1 in a 50 mM Na؉ solution, whereas the truncated protein binds to single-stranded DNA (ssDNA) with a much higher affinity of (7.8 ؎ 0.9) ؋ 107 M؊1 under the same solution conditions
Because the DNA is stretched during the single molecule experiment, measurements can be obtained under solution conditions that would allow the protein-DNA complex to aggregate in a bulk solution experiment

Summary

Introduction

Optical tweezers have been used extensively for studying the biomechanical properties of single DNA molecules by stretching the molecules and measuring the required force for a given extension under various conditions [1,2,3,4,5,6].

Results

Conclusion