Highlights d A single chromatin substrate is torsionally softer than a braided substrate d Thus, DNA supercoiling should primarily partition to the front of a replisome d Yeast topo II shows a strong preference for a single chromatin substrate d Thus, torsional mechanics and topoisomerase activity coordinate synergistically

The E. coli single-stranded DNA binding (SSB) protein interacts with at least 17 different proteins, known as SSB-interacting proteins (SIPs), during DNA replication, repair, and recombination. The E. coli RecO protein is a recombination mediator protein functioning in complex with RecF and RecR proteins in the RecF pathway of homologous recombination. RecO has been shown to interact with the last 9 amino acids of the intrinsically disordered C-terminal tails of SSB (SSB-Ct). Although structures of RecOR complexes from organisms, such as D. Radiodurans and T. Tencongensis, have been determined, they differ in stoichiometry. Furthermore, structures of the E. coli RecOR complex are not yet available. We therefore investigated the assembly states of RecO and RecR, and RecOR complexes using analytical ultracentrifugation. We find that E. coli RecO is a stable monomer. Although E. coli RecR had previously been reported to form a dimer, we find that it is in a pH-dependent dimer-tetramer equilibrium. We also find that only the RecR tetramer and not the dimer binds RecO and that the SSB-C-terminal peptide destabilizes the RecOR 4 complex (supported by NIH GM030498 to TML).