Imaging and energetics of single SSB-ssDNA molecules reveal intramolecular condensation and insight into RecOR function

Abstract

Escherichia coli single-stranded DNA (ssDNA) binding protein (SSB) is the defining bacterial member of ssDNA binding proteins essential for DNA maintenance. SSB binds ssDNA with a variable footprint of ∼30–70 nucleotides, reflecting partial or full wrapping of ssDNA around a tetramer of SSB. We directly imaged single molecules of SSB-coated ssDNA using total internal reflection fluorescence (TIRF) microscopy and observed intramolecular condensation of nucleoprotein complexes exceeding expectations based on simple wrapping transitions. We further examined this unexpected property by single-molecule force spectroscopy using magnetic tweezers. In conditions favoring complete wrapping, SSB engages in long-range reversible intramolecular interactions resulting in condensation of the SSB-ssDNA complex. RecO and RecOR, which interact with SSB, further condensed the complex. Our data support the idea that RecOR--and possibly other SSB-interacting proteins—function(s) in part to alter long-range, macroscopic interactions between or throughout nucleoprotein complexes by microscopically altering wrapping and bridging distant sites.

DOI: http://dx.doi.org/10.7554/eLife.08646.001

eLife digest

Chromosomes consist of two strands of DNA that are intertwined as a helix. These strands can peal apart to form single-stranded DNA before the DNA is copied and for other processes in cells. Single-stranded DNA can also form if double-stranded DNA is damaged by harmful radiation or chemicals so that only one strand can be copied or when the damaged strand is selectively degraded by enzymes during the course of repair.

Proteins called single-stranded binding proteins (or SSBs for short) bind to single-stranded DNA molecules to protect them. A molecule of single-stranded DNA wraps around a group of four SSB proteins (known as a tetramer). The degree to which DNA is wrapped around the SSB tetramer depends on the environmental conditions. For example, in the presence of high levels of salt—which is typical inside cells – single-stranded DNA wraps around all four subunits of the SSB. However, at lower salt levels, the DNA only wraps around some of the units in the SSB tetramer.

A process called recombination can repair breaks in DNA. During this process, a broken DNA molecule that contains single-stranded DNA can pair with a matching (or complementary) strand from an intact double-stranded DNA molecule that carries an identical genetic sequence. A protein called RecO helps to anneal two complementary DNA strands together with the help of the RecR protein. However, for RecR and RecO to achieve this task, they need to work together with the resident SSB proteins that occupy single-stranded DNA. How they find matching sequences when SSB proteins are in the way is not clear.

Bell et al. used techniques called TIRF microscopy and single-molecule force spectroscopy to directly observe how SSB from the bacterium E. coli binds to and coats individual molecules of single-stranded DNA. The experiments show that when the levels of salt increase, single-stranded DNA that is coated with SSB proteins becomes compacted and the length of the DNA molecules decreases, a process referred to as ‘intramolecular condensation’. Bell et al. found that condensation occurred because two SSB tetramers that are associated with different regions of the single-stranded DNA interact to form stable ‘octamers’.

In the presence of RecO and RecR, the single-stranded DNA compacted even further. Bell et al. propose that these recombination proteins act as a scaffold to bring together distant partner sites of single-stranded DNA. This condensation allows two DNA sequences that can be far apart in the cell to find one another more quickly. The next challenge is to understand how the matching regions of single-stranded DNA are identified, and what causes the SSBs to move to allow other repair proteins to gain access to the DNA.

DOI: http://dx.doi.org/10.7554/eLife.08646.002