Mechanism for the TtDnaA-Tt-oriC cooperative interaction at high temperature and duplex opening at an unusual AT-rich region in Thermoanaerobacter tengcongensis.
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 requires (i) at least two neighboring DnaA boxes, (ii) the specific feature of the TtDnaA Domain IV and (iii) the self-oligomerization 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 degrees C, but not at 37 degrees 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 oligomerization 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.
Pei
Huadong
H
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, PR China.
Liu
Jingfang
J
Li
Jie
J
Guo
Aobo
A
Zhou
Jian
J
Xiang
Hua
H
D020856:AT Rich Sequence; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D051738:Origin Recognition Complex; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D018741:Replication Origin; D013696:Temperature; D046968:Thermoanaerobacter / Q000235:genetics*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; C066312:DnaA protein, Bacteria; C114650:OriC chromosomal replication origin; D051738:Origin Recognition Complex; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate