Single Molecule Imaging of Tid1/Rdh54, a Rad54 Homolog That Translocates on Duplex DNA and Can Disrupt Joint Molecules

Amitabh V Nimonkar,Ichiro Amitani,Ronald J Baskin,Stephen C Kowalczykowski

doi:10.1074/jbc.m704767200

Amitabh V Nimonkar, Ichiro Amitani + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m704767200

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Abstract

The Saccharomyces cerevisiae Tid1 protein is important for the recombinational repair of double-stranded DNA breaks during meiosis. Tid1 is a member of Swi2/Snf2 family of chromatin remodeling proteins and shares homology with Rad54. Members of this family hydrolyze ATP and promote 1) chromatin remodeling, 2) DNA topology alterations, and 3) displacement of proteins from DNA. All of these activities are presumed to require translocation of the protein on DNA. Here we use single-molecule visualization to provide direct evidence for the ability of Tid1 to translocate on DNA. Tid1 translocation is ATP-dependent, and the velocities are broadly distributed, with the average being 84 +/- 39 base pairs/s. Translocation is processive, with the average molecule traveling approximately 10,000 base pairs before pausing or dissociating. Many molecules display simple monotonic unidirectional translocation, but the majority display complex translocation behavior comprising intermittent pauses, direction reversals, and velocity changes. Finally, we demonstrate that translocation by Tid1 on DNA can result in disruption of three-stranded DNA structures. The ability of Tid1 translocation to clear DNA of proteins and to migrate recombination intermediates may be of critical importance for DNA repair and chromosome dynamics.

Highlights

The TID1 (Two-hybrid interacting with Dmc1), [1] gene product, known as RDH54 (Rad homolog 54 ), is required for mitotic as well as meiotic recombination [2,3]
It is speculated that Tid1 primarily cooperates with Dmc1 to promote inter-homolog DNA strand exchange, whereas Rad54 would function with Rad51 to promote inter-sister DNA strand exchange [3,10,11]
Our study shows that Tid1 can ‘unwind’ three-stranded DNA structures by virtue of its ability to translocate on DNA, thereby establishing that Tid1 can act on intermediates of recombination

Summary

Introduction

The TID1 (Two-hybrid interacting with Dmc1), [1] gene product, known as RDH54 (Rad homolog 54 ), is required for mitotic as well as meiotic recombination [2,3]. Single-molecule Tid1 translocation assay − The FITC-Tid1-DNA-bead complex was introduced into the first channel of a multi channel flow cell. Translocation of Tid1 along dsDNA was initiated by moving the trapped FITC-Tid1-DNA-bead complex to the second channel of the flow cell containing a degassed solution of 40 mM Tris-acetate, pH 8.2, 30 mM dithiothreitol, 15% sucrose, 2 mM magnesium acetate and 1 mM ATP.

Results

Conclusion