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Contents

Introduction

Replication Terminator Protein (RTP) from Bacillus Subtilis is a protein of current scientific investigation in terms of its ability to bind DNA, its symmetric and asymmetric nature, and the mechanism upon which it terminates DNA replication. Belonging to a group of Replication Terminator Proteins that are commonly found in prokaryotes (in particular within the Bacillaceae family)[1], RTP is often compared to another protein with similar intracellular function, Termination Utilisation Sequence (Tus) from E. coli. RTP has been shown to exist in both symmetric (in solution and when bound to palindromic DNA sequences) and asymmetric states (when bound to native DNA). The structure of RTP has been shown to be integral to it's function. RTP must be able to bind DNA (and therefore must be positively charged) and bind asymetrically (despite it being a homomeric dimer) in order to specifically block DNA replication from one direction.

The Structure of RTP

Structure of RTP (PDB entry 1bm9)

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RTP binding to DNA

RTP bound to symmetric DNA (PDB entry 1f4k)

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The Asymetric binding

RTP bound to native DNA (PDB entry 2efw)

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Termination Mechanism

As previously noted the role of RTP is to terminate replication of the bacterial chromosome. It was originally assumed that the role of RTP was simply to arrest the replication fork allowing the DNA to cleanly separate [5]. The proposed mechanism noted that the replication fork is only able to disrupt the RTP/Ter interaction when approaching the A-site/"blocking face". The directionality of the Ter sites (ie. the orientation of A site vs B site) will determine from which direction replication will be arrested. However recent research has indicated a more complex mechanism involving interactions between bound RTP and the replication fork helicase. The results of this research have confirmed a RTP/DnaB interaction in vivo, further suggesting this interaction plays an important role in replication fork arrest [6]. This has lead to the development of a new helicase-specific model involving protein-protein interactions between the replication fork helicase and RTP protein which arrests the replication fork when it approaches from the appropriate direction [7]. This evidence alows us to move from a simple "fork arrest model" to a more complex understanding of termination.

Further Directions

RTP is frequently compared to Termination Utilisation Sequence (Tus) from E. coli. These two proteins display similar intracellular function with binding to Ter sites resulting in replication termination, despite the significant lack of identity and similarity between them (22% identity, 44% similarity) (Ref). Structurally these proteins differ as Tus has been demonstrated to be a monomer and an additional 300kbp larger than RTP. Investigations in to their comparative function have shown that the substitution of RTP for Tus in the E.coli system, will demonstrate no phenotypic difference, and hence share the same function as replication terminators. However the question still remains to be answered how can two structurally different proteins give rise to the same intracellular function. Hopefully, further investigations will be able to shed more light as to how RTP and Tus, from B. subtilis and E. coli respecively, arrest the replication fork mechanism.

References

  1. R.D. Finn, J. Mistry, J. Tate, P. Coggill, A. Heger, J.E. Pollington, O.L. Gavin, P. Gunesekaran, G. Ceric, K. Forslund, L. Holm, E.L. Sonnhammer, S.R. Eddy, A. Bateman The Pfam protein families database [1] Nucleic Acids Research (2010) Database Issue 38:D211-222
  2. 2.0 2.1 2.2 2.3 2.4 Bussiere DE, Bastia D, White SW. Crystal structure of the replication terminator protein from B. subtilis at 2.6 A. Cell. 1995 Feb 24;80(4):651-60. PMID:7867072
  3. 3.0 3.1 Vivian JP, Porter CJ, Wilce JA, Wilce MC. An asymmetric structure of the Bacillus subtilis replication terminator protein in complex with DNA. J Mol Biol. 2007 Jul 13;370(3):481-91. Epub 2007 Mar 2. PMID:17521668 doi:S0022-2836(07)00259-8
  4. Wilce JA, Vivian JP, Hastings AF, Otting G, Folmer RH, Duggin IG, Wake RG, Wilce MC. Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest. Nat Struct Biol. 2001 Mar;8(3):206-10. PMID:11224562 doi:10.1038/84934
  5. Wake RG. Replication fork arrest and termination of chromosome replication in Bacillus subtilis. FEMS Microbiol Lett. 1997 Aug 15;153(2):247-54. PMID:9271849
  6. Gautam A, Bastia D. A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control. J Biol Chem. 2001 Mar 23;276(12):8771-7. Epub 2000 Dec 21. PMID:11124956 doi:10.1074/jbc.M009538200
  7. Kaplan DL, Bastia D. Mechanisms of polar arrest of a replication fork. Mol Microbiol. 2009 Apr;72(2):279-85. Epub 2009 Mar 4. PMID:19298368 doi:10.1111/j.1365-2958.2009.06656.x

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