Structural Studies of Rolling Circle Replication Initiator Proteins

Abstract

pT181 family plasmids replicate by a rolling-circle mechanism. This is initiated by a plasmid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The replication origin is nicked by Rep, which binds covalently to one DNA strand via an active site tyrosine, initiating rolling circle replication and religating the strand at the end of the cycle. Rep proteins also associate with PcrA helicase to form a highly processive complex. We have determined the structure of the Rep protein from cryptic plasmid pSTK1 of Geobacillus stearothermophilus (Gst), and several variants of RepD from Staphylococcus aureus (Sau), representing the first structural information on this class of initiators.Cloning and expression of a construct derived from the from pSTK1 Rep yielded a product that relaxed plasmid substrates encoding an inverted repeat sequence from pSTK1, which resembles the replication origin of the pT181 family, and activated the cognate Gst PcrA helicase. The crystal structure for the 31 kDa fragment of Gst Rep has been solved at 2.3 A, showing a novel, ring-shaped dimer with a 20A diameter pore. The inner surface is formed by an 18-stranded β-sheet, while the outer surface is decorated with 18 α-helices. The protein has a novel fold, but the extended sheet exhibits similarities to that in TATA-binding protein (TBP). The active site Tyr179 residues, one from each subunit, lie 26 A apart across the pore, with a nearby catalytic magnesium ion co-ordinated by three carboxylate side-chains.Crystal structures for the Sau Rep variants RepDE, RepDN and RepDC have been solved by molecular repacement using the Gst Rep as a model, and show similar structural features. The implications for the mechanism of rolling circle replication will be discussed in the light of extensive functional data available for Sau RepD.