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Category: Microbial Genetics and Molecular Biology
Participating Elements in the Replication of Iteron-Containing Plasmids, Page 1 of 2
< Previous page | Next page > /docserver/preview/fulltext/10.1128/9781555817732/9781555812652_Chap02-1.gif /docserver/preview/fulltext/10.1128/9781555817732/9781555812652_Chap02-2.gifAbstract:
The presence of Rep-binding iterons is a hallmark, not only of many prokaryotic plasmid orfs but of chromosomal, viral (phage), and eukaryotic ori’s(5, 75, 92) as well. This chapter focuses primarily on the prokaryotic plasmid members of the Rep/iteron family, which is referred to as iteron-containing plasmids (ICPs). Several extensive reviews on a variety of aspects of plasmid biology have been written, and this review serves as an extension of the earlier works. Sequence conservation in the adjacent major and minor grooves has been demonstrated for iterons from prokaryotic and eukaryotic ori’s. It is likely, then, that a diverse set of Rep proteins can utilize the intrinsic instability of specific base pairs within iterons to bind DNA and facilitate its melting. The chapter discusses what appeared to be a rather well-understood system, λdv. It exemplifies the autorepressor model originally developed to explain the control of Escherichia coli chromosomal replication, and discusses some of the additional DNA sequences and host proteins that likely contribute to the regulation of ICPs. The chapter discusses molecular mechanism of ori activation, and focuses on Rep/iteron-mediated replication of minimal ICPs in E. coli. The contributions of plasmids to the development of modern molecular biology were astutely articulated a decade ago. Currently, the need for ongoing and vigorous support of basic and applied research on plasmids continues, with an imperative to solve practical issues of fundamental societal importance. A prime example is the emergence and alarming spread of bacterial resistance to multiple antibiotics.
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Schematics of ICP replication initiation. (A) The Rep protein (shaded ovals |R|) is produced from the rep gene (gray rectangle). Rep hinds in an orderly fashion to the iterons (hlack rectangle) and stimulates DNA replication (plus sign). Certain host proteins (examples in the gray box) will interact in solution with the Rep protein while others may interact with Rep bound to DNA. Host proteins also exert effects by directly binding to the DNA at multiple sites within the origin of replication (on). (B) A closer view of the protein-protein interactions model within the ori is depicted. Rep protein will interact (curved arrows) with host proteins that bind near the iterons or at distant sites (e.g., DnaA, black oval |A|). The distant sites can be brought closer by the DNA-bending ability of proteins like IHF (I), thus promoting protein interactions at different levels (Rep/host, host/host, and Rep/Rep).
Schematics of ICP replication initiation. (A) The Rep protein (shaded ovals |R|) is produced from the rep gene (gray rectangle). Rep hinds in an orderly fashion to the iterons (hlack rectangle) and stimulates DNA replication (plus sign). Certain host proteins (examples in the gray box) will interact in solution with the Rep protein while others may interact with Rep bound to DNA. Host proteins also exert effects by directly binding to the DNA at multiple sites within the origin of replication (on). (B) A closer view of the protein-protein interactions model within the ori is depicted. Rep protein will interact (curved arrows) with host proteins that bind near the iterons or at distant sites (e.g., DnaA, black oval |A|). The distant sites can be brought closer by the DNA-bending ability of proteins like IHF (I), thus promoting protein interactions at different levels (Rep/host, host/host, and Rep/Rep).
Regulation models for ICP replication. ( 1 ) Rep dimers are able to bind to the operator/promoter region (Op/Pr) and negatively regulate Rep production (minus sign). ( 2 ) Monomers of the replication initiator activate origin (ori) replication (plus sign). Panels 3 to 5 represent variations of the “handcuffing model.” ( 3 ) Two layers of monomers bound to the iterons of two individual plasmids are able to bring two ori's together (curved small arrows), shutting down replication of both (minus sign). ( 4 ) Dimers of the Rep protein bridge two plasmids by associations with monomers bound to iterons. ( 5 ) A single layer of dimers binds the iterons of two ori's, applicable only for those systems where dimers can directly bind iterons. ( 6 ) The “titration model” in which the presence of an extra set of iterons from a second plasmid could titrate the activator form of the Rep protein (monomers), leading to the depletion of Rep and thereby inactivating the ori.
Regulation models for ICP replication. ( 1 ) Rep dimers are able to bind to the operator/promoter region (Op/Pr) and negatively regulate Rep production (minus sign). ( 2 ) Monomers of the replication initiator activate origin (ori) replication (plus sign). Panels 3 to 5 represent variations of the “handcuffing model.” ( 3 ) Two layers of monomers bound to the iterons of two individual plasmids are able to bring two ori's together (curved small arrows), shutting down replication of both (minus sign). ( 4 ) Dimers of the Rep protein bridge two plasmids by associations with monomers bound to iterons. ( 5 ) A single layer of dimers binds the iterons of two ori's, applicable only for those systems where dimers can directly bind iterons. ( 6 ) The “titration model” in which the presence of an extra set of iterons from a second plasmid could titrate the activator form of the Rep protein (monomers), leading to the depletion of Rep and thereby inactivating the ori.
Replication steps—a model. The replication initiator protein (Rep) recognizes the origin of replication (or/) and induces a conformational change in the plasmid (e.g., DNA bending). Then Rep protein, with or without host proteins engaging their binding sites (IHF/HU, DnaA), triggers strand separation in an A+T-rich segment of the DNA. This single-stranded region is then targeted for the loading of DNA helicase and primase. DNA helicase will further unwind the DNA helix while primase will start synthesizing short RNA molecules, which serve as primers for the initiation of DNA synthesis by “sliding” DNA polymerase.
Replication steps—a model. The replication initiator protein (Rep) recognizes the origin of replication (or/) and induces a conformational change in the plasmid (e.g., DNA bending). Then Rep protein, with or without host proteins engaging their binding sites (IHF/HU, DnaA), triggers strand separation in an A+T-rich segment of the DNA. This single-stranded region is then targeted for the loading of DNA helicase and primase. DNA helicase will further unwind the DNA helix while primase will start synthesizing short RNA molecules, which serve as primers for the initiation of DNA synthesis by “sliding” DNA polymerase.