Albany 2013: Book of Abstracts
June 11-15 2013
©Adenine Press (2012)
Structural Studies of Rolling Circle Replication Initiator Proteins
Plasmids of the pT181 family replicate by a rolling-circle mechanism. The process is initiated by a plasmid-encoded Rep initiator protein, which has sequence-specific DNA nicking and religation activity. The plasmid 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 the designated 269 aa Rep product from pSTK1 failed to yield soluble, active protein. However, expression from an arbitrary point upstream yielded an elongated product capable of relaxing plasmid substrates encoding an inverted repeat sequence from pSTK1 which resembles the replication origin of the pT181 family. Both this product and a 31 kDa proteolytically derived fragment omitting the C-terminus additionally display activation of the cognate Gst PcrA helicase, but not that of S. aureus. The crystal structure of the 31 kDa fragment of Gst Rep has been solved at 2.3 Å resolution, showing an unusual ring-shaped dimer with a 20Å diameter pore. The inner surface of the ring is largely formed by an 18-stranded β-sheet, while the outer surface is decorated with 18 α-helices. The protein represents a novel fold, however, the extended sheet does exhibit some similarities to those observed in both TATA-binding protein and transcription factor IID. The active site Tyr179 residues, one from each subunit, lie 26 Å apart across the pore, with a nearby catalytic magnesium ion co-ordinated by three carboxylate side-chains.
Crystal structures for the Sau Rep variants termed 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.
Stephen B. Carr1
1Research Complex at Harwell