Structural features of the initiator of replication protein RepB encoded by the promiscuous plasmid pMV158 Jose ´ A. Ruiz-Maso ´ a , Consuelo Lo ´pez-Zumel b , Margarita Mene ´ndez b , Manuel Espinosa a, * , Gloria del Solar a a Centro de Investigaciones Biolo ´gicas, Consejo Superior de Investigaciones Cientificas, Vela ´zquez, 144, E-28006 Madrid, Spain b Instituto de Quı ´mica Fı ´sica Rocasolano, Serrano 119, Consejo Superior de Investigaciones Cientı ´ficas, Spain Received 30 June 2003; accepted 23 September 2003 Abstract The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% a-helices and 20% h-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 jC, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage. D 2003 Elsevier B.V. All rights reserved. Keywords: Initiator of replication protein; Conformational change; Circular dichroism; Analytical ultracentrifugation 1. Introduction Bacterial plasmids usually initiate replication by a plas- mid-encoded protein, generically termed Rep, which, in many cases, is rate-limiting. At least three different mech- anisms of plasmid replication can be considered, namely the theta type, the strand displacement, and the rolling circle (RC) mode [1]. There are more than 200 characterized small plasmids that use the RC-replication mechanism [2]. They have been isolated from a variety of bacteria and archaea, and have been grouped into 17 different families by M. Osborn http://www.essex.ac.uk/bs/staff/osborn/DPR _ home.htm). The plasmid RC mode of replication can be divided into various stages, as follows (reviewed in Refs. [1–3]). The Rep-initiator recognizes the plasmid double- strand origin of replication (dso) on a supercoiled DNA substrate. Upon binding to the bind region, the Rep initiator protein exerts a nucleophilic attack, mediated by a Tyr residue, on the phosphodiester bond of a specific dinucleotide within the dso. Nicking results in a covalent Rep-DNA adduct in which the 3V -end of the cleaved DNA is free whereas the 5V -end is blocked by a phosphotyrosine bond [4]. Next, the nick is extended by the action of host- encoded proteins, such as the PcrA helicase, DNA polymer- ase I, and DNA polymerase III, whereas the single-stranded DNA that is being ‘‘peeled-off’’ would be covered by the host-encoded single-stranded DNA binding protein [3]. Finally, a single-stranded DNA intermediate is generated, this type of molecules constituting the hallmark of the RC- replicating plasmids. Upon recognition of a DNA region on the single-stranded intermediate, the host-encoded RNA polymerase synthesizes a small RNA primer which is later elongated by the DNA polymerases [5], thus completing one round of replication. The RC-replication mechanism has been studied in some depth at least for some plasmids (reviewed in Ref. [3]), and the biochemistry of the Rep-mediated cleavage of DNA has been characterized for the Rep proteins of the staphylococ- cal plasmids pT181 [6,7] and pC221 [8] and of the strep- tococcal plasmid pMV158 [9,10]. In the case of the latter 1570-9639/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bbapap.2003.09.010 Abbreviations: bp, base pair; CD, circular dichroism; PCR, polymerase chain reaction; RC, rolling circle * Corresponding author. Tel.: +34-9156-11800; fax: +34-9156-27518. E-mail address: mespinosa@cib.csic.es (M. Espinosa). www.bba-direct.com Biochimica et Biophysica Acta 1696 (2004) 113– 119