Plasmid Rolling-Circle Replication

Abstract

15 16 17 18 19 20 1. General aspects 21 2. The double-strand origin 22 3. The replication initiator Rep proteins 23 4. Control of rolling-circle replication24 4.1. Mechanisms for regulating synthesis of the Rep protein25 4.2. Mechanisms that restrict the use of Rep molecules to a single replication event 26 5. The single-strand origin 27 6. Host proteins involved in rolling-circle replication 28 6.1. Participation of DNA and RNA polymerases 29 6.2. Role of SF1 helicases and their interaction with the plasmid-encoded Rep initiator 30 31 SUMMARY 1Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a 2 crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with 3 the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication 4 mechanisms offer divers solutions to this problem. Rolling-circle replication (RCR) is a mechanism 5 adopted by certain plasmids, among other genetic elements, that represents one of the simplest 6 initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to 7 generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. 8All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, 9 lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. 10RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been 11 described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the 12 RCR plasmids lifestyle with emphasis on their characteristic traits, promiscuity, stability, utility as 13 vectors, etc. While RCR is one of the better characterized plasmid replication mechanism, there are 14 still many questions left unanswered, which will be pointed out along this review.The main features that characterize rolling-circle replication (RCR; see Fig. 1A) derive from its singular 18 initiation mechanism, which relies on the sequence-specific cleavage, at the nick site of the double-19 strand origin (dso), of one of the parental DNA strands by an initiator Rep protein. This cleavage 20 generates a 3'-OH end that allows the host DNA polymerases to initiate the leading strand replication. 21Therefore, the RCR initiation circumvents the synthesis of a primer RNA that is required in all other 22 modes of replication of circular double-stranded (ds) DNA. Elongation of the leading strand takes 23 place as the parental double helix is unwound by a host DNA helicase and the cleaved non-template 24 strand is covered with the single-stranded DNA binding (SSB) protein. Since the nascent DNA is 25 covalently attached to the parental DNA, termination of a round of leading-strand replication implies a 26 new cleavage event at the reconstituted nick site. This reaction is assumed to be catalyzed by the 27 same Rep molecule that carried out the initiation cleavag...

Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158

“…The OD thus provides a rigid scaffold for the N- and C-terminal domains that need a certain level of conformational flexibility important for function 42 . In addition, we show that an OBD-OD hinge region may exist in viral Reps, which would confer the ability to process the DNA appropriately by facilitating the recognition of a variety of different DNA structures and topologies 15 43 . Moreover, severe distortion of the DNA might facilitate simultaneous binding of a Rep protein to all distant recognition sites in the origin DNA.…”

Rolling‐circle amplification of centromeric Helitrons in plant genomes

“…In addition, unlike most eukaryotic transposons and like prokaryotic RC transposons, Helitrons lacked terminal inverted repeats and target site duplications. RCR has been demonstrated for various prokaryotic phages and plasmids (Saleem, ; Ruiz‐Masó et al ., ). The prokaryotic initiator Rep protein plays a key role in RCR and the RepHel protein is postulated to play a similar role (Kapitonov and Jurka, ).…”

Structural analysis of a replication protein encoded by a plasmid isolated from a multiple sclerosis patient

“…Within prokaryotes, Rep plays a central role in maintaining the plasmid copy number, as reported for the F plasmid in Escherichia coli (Kline, 1985). This regulation is also critical for the replication of plasmidderived, bacteriophage-like or virus-like DNA genomes (Ruiz-Masó et al, 2015). Reps are essential for the replication of multidrug-resistant bacteria in humans (Schumacher et al, 2014) and studies have suggested that Reps have a role in transmissible amyloid proteinopathy (Molina-García et al, 2018;Giraldo et al, 2016Giraldo et al, , 2011.…”