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IntroductionIntroduction
Replication Termination in Escherichia coli and Bacillus subtilisReplication Termination in Escherichia coli and Bacillus subtilis
The replication of chromosomal DNA in most bacterial species occurs through a bidirectional mechanism, whereby two replication forks derived from the same origin of replication travel in opposite directions.[1] This gives rise to the characteristic theta-shaped structure as the nascent DNA loop connecting the replication forks is generated. While the use of two replisomes, one at each fork, accelerates replication, the phase of termination must be carefully coordinated. Specific sequences known as Ter sites lie just beyond the halfway point for each replisome. These halt the advance of replication forks in a direction-specific manner, and thereby form a replication fork trap. As a result, each replisome can traverse only slightly more than half of the DNA before it is arrested. Together, the two sets of Ter sites define the terminus region where replication is terminated and the two forks fuse.
The function of the replication fork trap is enacted by the binding of termination proteins to Ter sites. In E. coli, this is done by termination utilisation substance (Tus), and the functionally corresponding protein in B. subtilis is replication termination protein (RTP). The importance of these replication fork traps is still a matter of debate, as their inactivation does not interfere greatly with cell cycle progression[2], nor does it affect the well-being of daughter cells following cell division. However, the fact that the structurally and sequentially dissimilar Tus and RTP proteins both bind to ill-conserved Ter sites to elicit the same biological function suggests an evolutionary pressure towards a termination mechanism that involves replication fork traps. It has been noted that the majority of genes in the B. subtilis genome have their promoters proximal to the origin of replication. Terminating replication before the replisome reaches the promoters may help the smooth running of transcriptional processes [3] [4].
During fork fusion, it is proposed that the fork arriving at the permissive face of the replication fork trap displaces the terminator protein and causes the disassembly of the replisome. A combination of helicase, toposiomerase, polymerase and ligase then fill the remaining gaps to make a chromosome dimer with the two full-length chromosomes joined. RecQ, topoisomerase III, single-strand binding protein (SSBP) and DNA polymerase I are said to play a role in this process [5]. Finally, site-specific recombination takes place at the dif site to produce two identical monomeric chromosomes [6].
The ProteinsThe Proteins
RTPRTP
In B. subtilis, Ter sites are 30 bp in length with two imperfect inverted 16 bp repeats overlapping at a TAT motif. The upstream portion of the Ter site is called TerA; and the downstream portion, TerB. The sequence differences between these cause the bound RTP dimers to bind with different affinity and generate an assymetric complex capable of halting the progression of the replication fork only if the B site is encountered first.The mechanism by which this is achieved is discussed below in relation to the structure of the .
The RTP DimerThe RTP Dimer
The structure of an RTP monomer bears greatest similarity to the "classic winged-helix" motif, in which 'wings' project from the loop between the final two β sheets of their compact αβααββ structure.[7] The two major variations from this theme are the absence of a β1 sheet (the corresponding region is instead termed the β1 loop), and the presence of a fourth elongate α-helix at the C-terminus, which facilitates dimerisation. Each of these secondary structural elements are indicated in this .
The association of α4 helices into an antiparellel coiled coil leads to dimerisation (left). The conformation is further stabilised by interhelical salt bridges outside this region, as well as an aromatic network on the inner surface which form a hydrophobic core. The phenylalanine and tryptophan residues that form part of this network are shown in the image (right).
The crystal structure of RTP in its unbound state was determined in 1995. A C110S mutant was then generated to prevent the aggregation of RTP through cysteine oxidation. With a very similar structure and almost no change in dimerisation and DNA-binding capacities, the mutant was the protein of choice for later studies.
DNA BindingDNA Binding
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13-15 residues contribute to the attachment of an RTP molecule to the Ter DNA site. Most of this is contributed by the basic residues of the , which lies in the major groove of DNA.
Herman's: When an RTP dimer binds to a TerA or TerB site, the basic residues of the a3 helix become positioned at the major groove and the beta ribbon rests at the minor groove. Non-specific ionic interactions between the N-terminus and the DNA backbone stabilise the complex.
It is known, with some certainty, that the RTP dimer adopts an asymmetric arrangement upon binding of the TerB site. In the C110S mutant complexed with the native TerB sequence (2EFW), the two subunits interact differently with the DNA bases to produce wing-up and wing-down conformations. These can be distinguished by the angle the a2 helix makes with the a3 helix. It is likely that asymmetry is also introduced when the RTP dimer binds to the TerA site but the crystal structure of this complex has not been solved?
Although the TerA site inherently has a lower binding affinity for RTP, as evident from the larger dissociation constant associated with the RTP-TerA complex, positive cooperativity from the binding of RTP to TerB facilitates the binding of RTP to TerA. It is proposed that RTP bends the DNA at the TerB site in a manner that favours RTP binding at TerA. The RTP dimer at TerB may also present a surface for stabilising interactions with the dimer at TerA. This is believed to happen through the b1 loop and b3 strand (need to standardise the names)
The asymmetry of the dimer is shown by the names 'wing up' and 'wing down'. It is measured by the angle between the a2 and a3 heices, as shown .
Replication Termination ActivityReplication Termination Activity
The consequence of these different conformations is most prominent in the position of the B1 sheet. This is evident in the Tyr33 residue, Space, which contact DNA only in the wing-down conformation.
Herman's: The asymmetric arrangement of the RTP dimer means that certain regions of the protein are accessible from one face only. In particular, Y33 in the wing-down monomer always comes in contact with the replication fork that is arrested. Of interest is that Y33 is found in a region that carries some similarity to DnaB, and this region is believed to interact with DnaB, likely in combination with the adjacent hydrophobic patch, in order to suppress its helicase activity.* At the moment, it is unclear why, but both the TerA and TerB sites must be occupied for full replication termination activity. (Reference??)
- 1bm9 paper
TusTus
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RoleRole
Tus is the 36 kDa protein responsible for termination of replication in Escherichia coli. The chromosome of E. coli contains a set of six polar Ter DNA sequences arranged such that three with the same directionality are located on either half of the chromosome. This generates what is termed a replication-fork trap, which prevents replication from occurring towards the origin. The Ter sites contain a 20 bp consensus element to which a monomer of Termination Utilisation Substance (Tus) binds to form a polar DNA-protein complex which halts the progression of the replicative machinery from one direction only. The fork arrest mechanism depends on the blocking of the helicase activity of DnaB, which is the first component of the replisome to encounter the Ter-Tus complex.
Space
Space
Space
Structural OverviewStructural Overview
The structure of the Tus protein was determined in complex with TerA by Kamada et al., and shown to be a previously undescribed backbone conformation (.). It is divided into two domains (amino and carboxy), in which α-helical regions of each are spanned by a central β-sandwich which contacts 13 bp of DNA duplex (#Indicate domains). Three helices within the amino domain (αI αII, αIII) form an antiparallel bundle aligned parallel to the DNA (#Helix bundle). Another two helices (αIV, αV) clamp the DNA phosphate backbone at the non-permissive end, and forms the cytosine-specific pocket containing the crucial residues for anti-helicase activity (#Phosphate clamp). The main DNA-binding domain however is the exposed side of the double β sheet layer which provides several base-specific interactions. This lies within the major groove and causes a conformational change in the DNA involving a deepening of the major groove, and an expansion of the minor one (#Sheet position).
space
space
space
DNA BindingDNA Binding
Tus is among the most stable monomeric, sequence-specific, double-stranded DNA-binding proteins. Three major sets of interactions contribute to this; (1) the phosphate clamp within the amino domain, (2) base-specific polar interactions by the β-sheet within the major groove, and (3) non-polar contacts within the carboxy domain.
1. Interaction between beta sheets (shown in green) and the major groove of DNA ().
Replication Termination ActivityReplication Termination Activity
Tus binds to a conserved cytosine residue which is not base paired . The interactions between residues of the Tus protein and this unpaired cytosine nucleotide are shown in more detail .
Its ability to do this depends on the conserved glutamate residue E49. "The crystal structure of the Tus-Ter lock shows that Glu49 of Tus makes a water-mediated hydrogen bond with the 50- phosphate of the displaced A(7) nucleotide residue, and it would thus be expected to be partially defective in formation of the locked species." This is shown
E49K does not affect DNA binding but does affect anti-helicase activity in the ‘trapped’ complex E49 makes an indirect hydrogen bond to the phosphate of the ‘displaced’ nucleotide.
The HistoryThe History
Summary of progression of understanding.