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Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

ABSTRACT: Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwinds DNA by the concerted actions of its three contiguous structural modules. The structure also shows that the functionally unknown PriCT domain, which forms a compact module, plays a central role in DNA unwinding. The conservation of the PriCT domain in the C termini of some archaeo-eukaryotic primases indicates that it probably plays a similar role in these proteins. Thus, this is the first report providing the structural basis for the functional importance of the conserved PriCT domain and also reveals a novel mechanism for DNA unwinding by a single protein.

SUBMITTER: Itou H

PROVIDER: S-EPMC4319026 | biostudies-literature | 2015 Feb

REPOSITORIES: biostudies-literature

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Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

Itou Hiroshi H Yagura Masaru M Shirakihara Yasuo Y Itoh Tateo T

The Journal of biological chemistry 20141223 6

Duplex DNA is generally unwound by protein oligomers prior to replication. The Rep protein of plasmid ColE2-P9 (34 kDa) is an essential initiator for plasmid DNA replication. This protein binds the replication origin (Ori) in a sequence-specific manner as a monomer and unwinds DNA. Here we present the crystal structure of the DNA-binding domain of Rep (E2Rep-DBD) in complex with Ori DNA. The structure unveils the basis for Ori-specific recognition by the E2Rep-DBD and also reveals that it unwind ...[more]

PMID: 25538245

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Project description: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.

Dataset Information

Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

Publications

Structural basis for replication origin unwinding by an initiator primase of plasmid ColE2-P9: duplex DNA unwinding by a single protein.

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