Abstract
DNA replication is tightly regulated to constrain the genetic material within strict spatiotemporal boundaries and copy numbers. Bacterial plasmids are autonomously replicating DNA molecules of much clinical, environmental and biotechnological interest. A mechanism used by plasmids to prevent over-replication is ‘handcuffing’, i.e. inactivating the replication origins in two DNA molecules by holding them together through a bridge built by a plasmid-encoded initiator protein (Rep). Besides being involved in handcuffing, the WH1 domain in the RepA protein assembles as amyloid fibres upon binding to DNA in vitro. The amyloid state in proteins is linked to specific human diseases, but determines selectable and epigenetically transmissible phenotypes in microorganisms. Here we have explored the connection between handcuffing and amyloidogenesis of full-length RepA. Using a monoclonal antibody specific for an amyloidogenic conformation of RepA-WH1, we have found that the handcuffed RepA assemblies, either reconstructed in vitro or in plasmids clustering at the bacterial nucleoid, are amyloidogenic. The replication-inhibitory RepA handcuff assembly is, to our knowledge, the first protein amyloid directly dealing with DNA. Built on an amyloid scaffold, bacterial plasmid handcuffs can bring a novel molecular solution to the universal problem of keeping control on DNA replication initiation.
Highlights
Amyotrophic lateral sclerosis to dialysis-related amyloidosis and type-II diabetes[18,19]
Dot-blot analysis of serial dilutions of the titration points for both types of DNA sequences revealed that samples including RepA-iteron complexes (Fig. 1b) were labelled with B3h7 up to higher dilutions than those with RepA-operator complexes (Fig. 1a) and, importantly, only at the titration points in which handcuffing complexes were evident in Electrophoretic mobility-shift assays (EMSA), whereas α -WH1 recognized both kinds of samples to a similar extent
The findings reported here integrate amyloids in the complex conformational transactions experienced by RepA protein in a key process for the biology of plasmid extrachromosomal mobile genetic elements in Gram-negative bacteria: i) DNA-bound dimers repress repA gene transcription; ii) DNA-bound monomers initiate DNA replication; and iii) by means of handcuffing, amyloidogenic oligomers inhibit post-replicative origin firing
Summary
Amyotrophic lateral sclerosis to dialysis-related amyloidosis and type-II diabetes[18,19]. Work performed on yeast prions[20] and bacterial biofilms[21] have clearly shown that amyloids can be functional, i.e. provide microorganisms with quickly selectable epigenetic, gain of function phenotypes[22]. We explore the link between regulation of pPS10 replication by RepA-mediated origin handcuffing and DNA-promoted RepA-WH1 amyloidosis. We have found that the regulatory RepA handcuffs are, to our notice, the first intracellular functional amyloids found in bacteria or involved in DNA replication
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