Uncoupling of Nucleotide Hydrolysis and Polymerization in the ParA Protein Superfamily Disrupts DNA Segregation Dynamics |
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Authors: | Aneta Dobruk-Serkowska Marisa Caccamo Fernando Rodríguez-Casta?eda Meiyi Wu Kerstyn Bryce Irene Ng Maria A Schumacher Daniela Barillà Finbarr Hayes |
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Institution: | From the ‡Faculty of Life Sciences and ;the §Manchester Institute of Biotechnology, The University of Manchester, Manchester M1 7DN, United Kingdom.;the ¶Department of Biology, University of York, York YO10 5DD, United Kingdom, and ;the ‖Department of Biochemistry, Duke University Medical Center, Durham, North Carolina 27710 |
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Abstract: | DNA segregation in bacteria is mediated most frequently by proteins of the ParA superfamily that transport DNA molecules attached via the segrosome nucleoprotein complex. Segregation is governed by a cycle of ATP-induced polymerization and subsequent depolymerization of the ParA factor. Here, we establish that hyperactive ATPase variants of the ParA homolog ParF display altered segrosome dynamics that block accurate DNA segregation. An arginine finger-like motif in the ParG centromere-binding factor augments ParF ATPase activity but is ineffective in stimulating nucleotide hydrolysis by the hyperactive proteins. Moreover, whereas polymerization of wild-type ParF is accelerated by ATP and inhibited by ADP, filamentation of the mutated proteins is blocked indiscriminately by nucleotides. The mutations affect a triplet of conserved residues that are situated neither in canonical nucleotide binding and hydrolysis motifs in the ParF tertiary structure nor at interfaces implicated in ParF polymerization. Instead the residues are involved in shaping the contours of the binding pocket so that nucleotide binding locks the mutant proteins into a configuration that is refractory to polymerization. Thus, the architecture of the pocket not only is crucial for optimal ATPase kinetics but also plays a key role in the polymerization dynamics of ParA proteins that drive DNA segregation ubiquitously in procaryotes. |
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Keywords: | ATPases Bacteria Escherichia coli Microbiology Molecular Biology Protein Self-assembly ParA ParF Partition Plasmid |
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