Interaction of the replication terminator protein of Bacillus subtilis with DNA probed by NMR spectroscopy

Termination of DNA replication in Bacillus subtilis involves the polar arrest of replication forks by a specific complex formed between the dimeric 29 kDa replication terminator protein (RTP) and DNA terminator sites. We have used NMR spectroscopy to probe the changes in ¹H-¹⁵N correlation spectra of a ¹⁵N-labelled RTP.C110S mutant upon the addition of a 21 base pair symmetrical DNA binding site. Assignment of the ¹H-¹⁵N correlations was achieved using a suite of triple resonance NMR experiments with ¹⁵N,¹³C,70% ²H enriched protein recorded at 800 MHz and using TROSY pulse sequences. Perturbations to ¹H-¹⁵N spectra revealed that the N-termini, α3-helices and several loops are affected by the binding interaction. An analysis of this data in light of the crystallographically determined apo- and DNA-bound forms of RTP.C110S revealed that the NMR spectral perturbations correlate more closely to protein structural changes upon complex formation rather than to interactions at the protein-DNA interface.