Recombination function and recombination kinetics of Escherichia coli single-stranded DNA-binding protein

Abstract

It is unknown whether the ssDNA-binding-protein (SSB) possesses the ability to catalyze DNA recombination. We investigated the recombination function of SSB and the recombination kinetics of Escherichia coli using a new transformation method with a modified double-layered plate. We found that SSB catalysed intermolecular recombination in vitro. Its intermolecular recombination rate versus substrate concentration or homologous sequence length fitted the Hill equation, and while the plasmid intramolecular recombination rate versus substrate concentration fitted a positively linear correlation, the dominant intermolecular recombination was a non-homologous recombination in vivo, similar to RecA. However, ssb-dependent recombination occurred later and at a lower recombination rate than the recA-dependent, probably because ssb expression was about two-fold lower than recA during the E. coli earlier growth stage. The affinity to substrate and the recombination efficiency of SSB was lower than RecA, whereas SSB enhanced the catalytic efficiency of RecA. Knocking out both recA and ssb led to loss of recombination. Our results confirmed that as SSB has the recombination function itself as an allosteric enzyme, recA-independent recombination in E. coli should be ssb-dependent. ssb-dependent recombination may be the third DNA double-strand break repair pathway, in addition to recA-dependent recombination and non-homologous end joining.