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.
Highlights
Single-stranded DNA binding protein (SSB) binds rapidly and avidly to ssDNA generated during the normal processes of DNA replication, recombination, and repair (Meyer and Laine, 1990)
SSBAF488-ssDNA nucleoprotein complexes were formed by first denaturing bacteriophage λ genomic dsDNA that had been biotinylated at the 3′-terminated ends using DNA polymerase (Figure 1A)
When the SSBAF488 was exchanged for unmodified wild type SSB, which has a higher affinity for ssDNA, the fluorescence rapidly decreased as SSBAF488 was displaced from the ssDNA (Figure 1F)
Summary
Single-stranded DNA (ssDNA) binding protein (SSB) binds rapidly and avidly to ssDNA generated during the normal processes of DNA replication, recombination, and repair (Meyer and Laine, 1990). The extent to which ssDNA is wrapped around a tetramer of SSB is often referred to as a binding mode, defined by the apparent site size or footprint (i.e. nucleotides bound per tetramer). These binding modes are sensitive to salt, temperature, pH, and binding density (Lohman and Ferrari, 1994). At higher more physiological salt concentrations, SSB binds in the fully wrapped binding mode and exhibits ‘limited’ cooperativity, where SSB forms dimers of tetramers (i.e. octamers) along the ssDNA (Bujalowski and Lohman, 1987; Lohman and Ferrari, 1994)
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