Mechanism for the TtDnaA-Tt-oriC cooperative interaction at high temperature and duplex opening at an unusual AT-rich region in Thermoanaerobacter tengcongensis

Abstract

Thermoanaerobacter tengcongensis is an anaerobic low-GC thermophilic bacterium. To further elucidate the replication initiation of chromosomal DNA at high temperature, the interaction between the replication initiator (TtDnaA) and the putative origin (Tt-oriC) in this thermophile was investigated. We found that efficient binding of TtDnaA to Tt-oriC at high temperature req uires (i) at least two neighboring DnaA boxes, (ii) the specific feature of the TtDnaA Domain IV and (iii) the selfoligomerization of TtDnaA. Replacement of the TtDnaA Domain IV by the counterpart of Escherichia coli DnaA or disruption of its oligomerization by amino acid mutations (W9A/L20S) abolished the oriC-binding activity of TtDnaA at 60°C, but not at 37°C. Moreover, ATP-TtDnaA, but not ADP-TtDnaA or the oligomerization-deficient mutants was able to unwind the Tt-oriC duplex. The minimal oriC required for this duplex opening in vitro was demonstrated to consist of DnaA boxes 1-8 and an unusual AT-rich region. Interestingly, although no typical ATP-DnaA box was found in this AT-rich region, it was exclusively bound by ATP-TtDnaA and acted as the duplex opening and replication-initiation site. Taken together, we propose that o ligomerization of ATP-DnaA and simultaneously binding of several DnaA boxes and/or AT-rich region may be generally required in replication initiation at high temperature. © 2007 The Author(s).