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<Structure load='2EWJ' size='500' frame='true' align='right' caption='Structure of the Tus-''Ter''B complex' scene='Insert optional scene name here' />
 
== Mechanisms of Polar Arrest ==
== Mechanisms of Polar Arrest ==

Revision as of 05:37, 20 May 2011

Contents

Termination sites in B.subtilis and E. coli

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Structures of the Replication Termination Proteins

RTP

Structure of RTP complexed with a native RTP B site

Drag the structure with the mouse to rotate

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Tus

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Mechanisms of Polar Arrest

RTP

The ability of RTP to induce polar fork arrest in B. subtilis poses a problem, namely, how does a symmetrical dimer block the replication fork? Early analysis of the B. subtilis ter sites revealed two RTP binding sites, a high affinity core site (B site) and a low affinity auxiliary site (A site)[1] . Mutagenic studies noted that generally contrahelicase activity was inversely correlated with binding affinity, though some mutant forms were also found that showed no loss in DNA binding affinity despite a partial or complete loss in contrahelicase activity [1] [2].

However, initial studies of RTP suggested that polar fork arrest was more complex than just DNA binding affinity. Separate mutagenesis studies showed that contrahelicase activity was dependent on dimer-dimer interactions between RTP molecules bound to the ter site [3]. Additionally, early studies of the protein structure predicted an asymmetrical complex would aid in the contrahelicase activity of RTP [4].

The issue of RTP and polar fork arrest was further complicated by Duggin et al. (2001) [5]. Using X-ray crystallography and NMR spectroscopy, the group found RTP formed a symmetric complex with a synthetic symmetric RTP B site (sRB). It was found that in the symmetrical form, extensive dimer interactions lead to an increase in DNA binding affinity. This lead to the Differential Binding Affinity (DBA) model of RTP where polar fork arrest activity was generated from the differences between binding the high affinity B site and low affinity A site within the ter sequence, due to the differences in contact bases between the two sites.

Soon after the publishment of Duggin et al. (2001) however, a series of experiments displayed the DBA model to be inadequate in explaining the polar fork arrest. Dixon et al. (2005) showed sRB to be a poor terminator sequence in vitro, and found no correlation between fork arrest activity and the affinity of the proximal binding site [6]. Furthermore, Duggin (2006) showed that fusing peptides to the C-terminus of RTP could eliminate fork arrest activity, independent of size of amino acid sequence. The fusion of the peptide did not affect any DNA binding sites, but rather an area of RTP that was proposed to contact the oncoming helicase [7].

Such lines of evidence lead to a revaluation of the RTP-ter structure. Porter et al. (2007) determined the crystal structure of RTP with a native RTP B binding site (nRB) [8]. The paper found that the RTP dimer formed an asymmetric complex with nRB due to differences in downstream base contacts with the α3 recognition helix of RTP, creating a “wing-up”/”wing-down” dimer. Modelling of the structure showed the asymmetric complex would allow for extensive protein interactions between RTP dimers, thus underlying the cooperative binding that is crucial for fork arrest. Taken together, these lines of evidence suggest that the polar fork arrest activity of RTP is dependent upon asymmetric contacts between RTP and the oncoming helicase, as mediated by the asymmetric interactions with the C-terminus [9].

Tus

PDB ID 2EWJ

Drag the structure with the mouse to rotate

Despite the polar fork arrest activity of the Tus-ter complex in E. coli, fundamental differences exist between it and RTP-ter system found in B. subtilis. A most basic difference is the fact Tus binds to ter as a monomer, rather than a dimer as is the case with RTP, and that this intrinsic asymmetry was likely to be the basis of Tus’ polar fork arrest activity [9]. Furthermore, the crystal structures of RTP and Tus show vastly different tertiary protein folds and DNA binding motifs [10].

Early studies of the Tus fork arrest mechanism lead to the formation of two separate models; the “clamp model” and the “interactions model” [11]. Structural analysis appeared to support the “clamp model”, showing Tus would bind tightly the DNA and impede sterically the replication fork with the αIV and αV helices [10]. However, some biochemical data seemed to indicate that a strong interaction occurred between Tus and the helicase. For instance, Bastia et al. (2001) displayed protein-protein interactions existed between Tus and DnaB using GST affinity chromatography and the yeast two-hybrid system. Furthermore, a series of mutants were identified with reduced Tus-DnaB interactions. These mutants were found to have substitutions within the L1 loop, and were shown to be deficient in fork arrest activity despite having higher DNA binding affinities [12]. However, much of the biochemical data is inconsistent, and Tus has been known to block fork arrest of unrelated helicases in a polar manner, both supporting the clamp model [11].

Cross et al. (2006) presented evidence for a modified clamp model [13]. Using a series of forked Tus-ter complexes, the group was able to show that fork progression from the non-permissive face leads to an increase in the stability of the complex. Further analysis of the forked complexes identified the C (6) residue in ter to be responsible for the locking via a base flipping mechanism. X-ray crystallography found the cytosine residue flipped in a binding pocket where it could hydrogen bond to Gly149, His 144 and Leu150 as well as forming hydrophobic interactions with Ile 79 and Phe 140. This “molecular mousetrap” was proposed to be the basis of fork arrest on the non-permissive face of Tus. Doubly forked complexes showed Tus was removed from the complex as DNA contacts were removed as the fork progressed on the permissive side, thus accounting for polarity.

However, recent evidence suggests specific interactions between Tus and the oncoming helicase may be the basis of polar fork arrest. Using a sliding helicase lacking strand separation activity, Bastia et al. (2008) showed Tus was able to block the sliding helicase on the non-permissive side even when the bases preceding C(6) were cross linked, and when the cytosine had been substituted. Hence, Tus was able to induce fork arrest without the base flipping mechanism. Furthermore, the group showed mutations in the L1 loop of Tus diminished its ability to arrest the sliding helicase [14].

Such evidence led to the establishment of a model for polar fork arrest as seen in Bastia and Kaplan (2009) [9]. In this mechanism, a helicase approaching from the non-permissive face of the Tus-ter complex specifically interacts with Tus, whilst also being sterically impeded. In this mechanism, the base-flipping lock is used a fail-safe to ensure fork arrest. At the same time, a helicase approaching from the permissive face would be able to dislodge Tus due to the lack of protein-protein interactions.

Comparison between the mechanism of RTP and Tus

It is important to note that there is a degree of similarity between the polar arrest mechanism of RTP and Tus, in so far that specific interactions with an oncoming helicase is required for its impediment. In both cases, the polarity is established by a structural asymmetry. The idea that the two proteins have some basic mechanistic similarity is supported by the fact that RTP can arrest fork progression in E. coli, as can Tus in B. subtilis, albeit with far less efficiently [9].

However, this asymmetry is accomplished in distinct ways; Tus is an inherently asymmetric monomer, whereas RTP takes on an asymmetric conformation once bound to DNA. RTP also lacks any specific base locking mechanism, as seen by the flipping of C (6) in the Tus-ter complex. The fact that such different proteins can have similar functions whilst sharing some basic similarities in mechanism supports the idea of independent, convergent evolution of the systems in E. coli and B. subtilis [10].


Future Researc: the dif site

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References

  1. ↑ 1.0 1.1 Bussiere DE, Bastia D. Termination of DNA replication of bacterial and plasmid chromosomes. Mol Microbiol. 1999 Mar;31(6):1611-8. PMID:10209736
  2. ↑ Pai KS, Bussiere DE, Wang F, Hutchison CA 3rd, White SW, Bastia D. The structure and function of the replication terminator protein of Bacillus subtilis: identification of the 'winged helix' DNA-binding domain. EMBO J. 1996 Jun 17;15(12):3164-73. PMID:8670817
  3. ↑ Manna AC, Pai KS, Bussiere DE, White SW, Bastia D. The dimer-dimer interaction surface of the replication terminator protein of Bacillus subtilis and termination of DNA replication. Proc Natl Acad Sci U S A. 1996 Apr 16;93(8):3253-8. PMID:8622923
  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
  5. ↑ 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
  6. ↑ Duggin IG, Matthews JM, Dixon NE, Wake RG, Mackay JP. A complex mechanism determines polarity of DNA replication fork arrest by the replication terminator complex of Bacillus subtilis. J Biol Chem. 2005 Apr 1;280(13):13105-13. Epub 2005 Jan 18. PMID:15657033 doi:10.1074/jbc.M414187200
  7. ↑ Duggin IG. DNA replication fork arrest by the Bacillus subtilis RTP-DNA complex involves a mechanism that is independent of the affinity of RTP-DNA binding. J Mol Biol. 2006 Aug 4;361(1):1-6. Epub 2006 Jun 21. PMID:16822523 doi:10.1016/j.jmb.2006.06.013
  8. ↑ 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
  9. ↑ 9.0 9.1 9.2 9.3 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
  10. ↑ 10.0 10.1 10.2 Kamada K, Horiuchi T, Ohsumi K, Shimamoto N, Morikawa K. Structure of a replication-terminator protein complexed with DNA. Nature. 1996 Oct 17;383(6601):598-603. PMID:8857533 doi:http://dx.doi.org/10.1038/383598a0
  11. ↑ 11.0 11.1 Neylon C, Kralicek AV, Hill TM, Dixon NE. Replication termination in Escherichia coli: structure and antihelicase activity of the Tus-Ter complex. Microbiol Mol Biol Rev. 2005 Sep;69(3):501-26. PMID:16148308 doi:10.1128/MMBR.69.3.501-526.2005
  12. ↑ Mulugu S, Potnis A, Shamsuzzaman, Taylor J, Alexander K, Bastia D. Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction. Proc Natl Acad Sci U S A. 2001 Aug 14;98(17):9569-74. Epub 2001 Aug 7. PMID:11493686 doi:10.1073/pnas.171065898
  13. ↑ Mulcair MD, Schaeffer PM, Oakley AJ, Cross HF, Neylon C, Hill TM, Dixon NE. A molecular mousetrap determines polarity of termination of DNA replication in E. coli. Cell. 2006 Jun 30;125(7):1309-19. PMID:16814717 doi:10.1016/j.cell.2006.04.040
  14. ↑ Bastia D, Zzaman S, Krings G, Saxena M, Peng X, Greenberg MM. Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase. Proc Natl Acad Sci U S A. 2008 Sep 2;105(35):12831-6. Epub 2008 Aug 15. PMID:18708526 doi:10.1073/pnas.0805898105

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