Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158

Ruiz-Masó, José A.; López-Zúmel, Consuelo; Menéndez, Margarita; Espinosa, Manuel; Solar, Gloria del

doi:10.1016/j.bbapap.2003.09.010

Cited by 13 publications

(19 citation statements)

References 28 publications

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“…RepB was obtained by a new method which yields high concentrations of pure protein (21). Analytical ultracentrifugation analyses of this protein preparation (not shown) revealed that RepB purified as a hexamer of identical subunits.…”

Section: Resultsmentioning

confidence: 99%

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Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

Ruiz-Masó

Espinosa

Solar

2007

Nucleic Acids Research

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Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

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Section: Resultsmentioning

confidence: 99%

“…Studier) was used as the host for purification of RepB. Plasmid pGEM repB 1, containing repB under the control of promoter ϕ10 of phage T7 (18,21), was employed.…”

Section: Methodsmentioning

confidence: 99%

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Interactions between the RepB initiator protein of plasmid pMV158 and two distant DNA regions within the origin of replication

Ruiz-Masó

Espinosa

Solar

2007

Nucleic Acids Research

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“…RepB is the first and to date the only published example of an atomic structure of a Rep protein from RCR plasmids or bacteriophages (3,90). Purified full-length native RepB behaves as a hexamer in solution, as observed in analytical ultracentrifugation assays (91), and is crystallized in the same oligomeric state, forming a toroidal homohexameric ring (90). Each RepB protomer comprises an N-terminal origin binding domain (OBD), which retains the DNA binding capabilities as well as the nuclease and strand-transfer activities of RepB, and a C-terminal oligomerization domain (OD) that forms a cylinder with a 6-fold symmetry in the hexamer (Fig.…”

Section: The Replication Initiator Rep Proteinsmentioning

confidence: 99%

Plasmid Rolling-Circle Replication

et al. 2015

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Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

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“…At least three different mechanisms of plasmid replication have been recognized, namely theta type, strand displacement, and rolling circle mode of replication (RCR), among which RCR is widespread in bacterial plasmids (8). Bacterial plasmids usually initiate replication by a plasmid-encoded protein, generically termed Rep (9).…”

Section: Introductionmentioning

confidence: 99%