The Arg Fingers of Key DnaA Protomers Are Oriented Inward of the Replication Origin oriC and Stimulate DnaA Subcomplexes in the Initiation Complex [Microbiology]

June 30th, 2015 by Noguchi, Y., Sakiyama, Y., Kawakami, H., Katayama, T.

ATP-DnaA binds to multiple DnaA boxes in the E. coli replication origin (oriC) and forms left-half and right-half subcomplexes that promote DNA unwinding and DnaB helicase loading. DnaA forms homo-oligomers in a head-to-tail manner via interactions between the bound ATP and Arg285 of the adjacent protomer. DnaA boxes R1 and R4 reside at the outer edges of the DnaA-binding region and have opposite orientations. In this study, roles for the protomers bound at R1 and R4 were elucidated using chimeric DnaA molecules that have alternative DNA binding-sequence specificity and chimeric oriC molecules bearing the alternative DnaA-binding sequence at R1 or R4. In vitro, protomers at R1 and R4 promoted initiation regardless of whether the bound nucleotide was ADP or ATP. Arg285 was shown to play an important role in the formation of subcomplexes that were active in oriC unwinding and DnaB loading. The results of in vivo analysis using the chimeric molecules were consistent with the in vitro data. Taken together, the data suggest a model in which DnaA subcomplexes form in symmetrically opposed orientations and in which the Arg285 fingers face inwards to mediate interactions with adjacent protomers. This mode is consistent with initiation regulation by ATP-DnaA and bidirectional loading of DnaB helicases.
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