Interactions between TrwB protein and DNA substrates with secondary structures

Abstract

TrwB is a DNA-dependent ATPase involved in DNA transport during bacterial conjugation. The protein presents structural similarity to hexameric molecular motors such as F1-ATPase, FtsK or ring helicases, suggesting that TrwB also operates as a motor, using energy released from ATP hydrolysis to pump ssDNA through its central channel. During this year, we have carried out an extensive analysis with various DNA substrates to determine the preferred substrate for TrwB. Oligonucleotides with G-rich sequences forming G4 DNA structures were the optimal substrates for TrwB ATPase activity. The protein bound with 100-fold higher affinity to G4 DNA than to ssDNA of the same sequence. Moreover, TrwB formed oligomeric protein complexes only with oligonucleotides presenting such a G-quadruplex DNA structure, consistent with stoichiometry of 6 TrwB monomers to G4 DNA, as demonstrated by gel filtration chromatography and analytical ultracentrifugation experiments. G-quadruplex structures are widespread in the genomes and are thought to play a biological function in transcriptional regulation. They form very stable structures that can obstruct DNA replication, requiring the action of specific helicases to resolve them. These observations are discussed in terms of a possible role for TrwB in resolving G4 secondary structures that arise during conjugative DNA processing.