Metal-Induced Stabilization and Activation of Plasmid Replication Initiator RepB

Abstract

Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin (dso) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn2+and the Mn2+-driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn2+ at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn2+ on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed.

Replication of Staphylococcal Resistance Plasmids

“…In contrast to the Rep_trans type initiators, the active Tyr residue in pMV158 RepB does not appear to form a stable covalent phosphodiester bond with the 5′-end of the cleaved strand ( Moscoso et al, 1995 ). However, a more labile RepB-DNA covalent adduct was observed after rapid treatment of cleavage reactions with SDS and proteinase K ( Ruiz-Masó et al, 2016 ), indicating that RepB inactivation after one round of leading strand synthesis might still occur through a similar mechanism.…”

Acidic pH Decreases the Endonuclease Activity of Initiator RepB and Increases the Stability of the Covalent RepB-DNA Intermediate while Has Only a Limited Effect on the Replication of Plasmid pMV158 in Lactococcus lactis

“…Recently, we succeeded in detecting a product arising from the RepB endonuclease activity at the nick site that corresponded to the 3′-fragment of a 3′-end fluorescently labeled substrate oligonucleotide and exhibited a decreased electrophoretic mobility relative to the corresponding protein-free DNA product on 20% PAA-urea gels. This slowly migrating product could only be observed when the temperature and time of incubation of the substrate DNA with either RepB 6 or OBD were reduced with respect to previously-reported conditions (from 60°C to ≤37°C and from 30 min to ≤15 min, respectively), and after treatment of the reaction products with 0.2% SDS and 0.1 mg/ml proteinase K ( Ruiz-Masó et al, 2016 ). The anomalous migration of the labeled reaction product (which actually appears as a set of 2–3 bands) was hypothesized to arise from a small protein polypeptide that remained bound to the 3′-fragment generated upon cleavage of the substrate oligonucleotide by the RepB 6 or OBD protein.…”

From Data Mining of Chitinophaga sp. Genome to Enzyme Discovery of a Hyperthermophilic Metallocarboxypeptidase

“…The metal which is physiologically relevant for M32 family is uncertain, as illustrated by the variety of metallic cations (Zn 2+ or Co 2+ ) found in the active site of the M32 superfamily of carboxypeptidases [38,65,66]. This scarcity of information on the preference of cations makes it difficult to discern whether the selection of the cation reflects a preference of the proteins or a greater availability of a specific metal in the cellular environment [75]. In this context, due to the fact that ChtCP showed a similar affinity between Co 2+ and Mn 2+ and is active in the presence of other metals (Mn 2+ , Mg 2+ , Co 2+ , Cd 2+ , Cr 3+ and Zn 2+ ), these features together make ChtCP very attractive in terms of its physiology when compared to other homologous proteins from M32 family, which are usually only active in the presence of Zn 2+ or Co 2+ .…”