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DNA stretching by bacterial initiators promotes replication origin opening

Abstract

Many replication initiators form higher-order oligomers that process host replication origins to promote replisome formation. In addition to dedicated duplex-DNA-binding domains, cellular initiators possess AAA+ (ATPases associated with various cellular activities) elements that drive functions ranging from protein assembly to origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been proposed to assist in single-stranded DNA formation during origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA opens replication origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal notable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic-acid-dependent, AAA+ systems.

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Figure 1: The ATPase pore of assembled DnaA binds ssDNA.
Figure 2: DnaA engages ssDNA in a manner similar to RecA.
Figure 3: DnaA extends ssDNA in solution.
Figure 4: DnaA directly melts duplex DNA.
Figure 5: Common DNA recognition strategies of AAA+ proteins.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

Coordinates have been deposited in the RSCB Protein Data Bank under the accession number 3R8F.

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Acknowledgements

We would like to thank K. Drlica, J. Keck, T. Murray and the Berger laboratory for helpful comments, and M. M. Cox for his contribution of RecA protein. This work was supported by the NIGMS (GM071747) and the National Institute of Health Molecular Biophysics Training Grant T32 GM008295.

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K.E.D. and J.M.B. designed the experiments, analysed the data and wrote the paper. Protein purification, crystallization and ssDNA binding assays were performed by K.C. and K.E.D. K.E.D. performed the other experiments.

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Correspondence to James M. Berger.

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Duderstadt, K., Chuang, K. & Berger, J. DNA stretching by bacterial initiators promotes replication origin opening. Nature 478, 209–213 (2011). https://doi.org/10.1038/nature10455

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