Bacterial Replication Termination

Replication Termination Protein (RTP), found in Bacillus subtilis, is a member of the ‘winged helix’ protein family, and terminates bacterial DNA replication by arresting the replication forks through interactions with DNA in a sequence specific manner [7]. RTP blocks the replication fork through contrahelicase activity; the ability to specifically inhibit the helicase replication machinery and has an additional role in arresting transcription [1][2]. In B. subtilis the bipartite ter sequence is overlapping, and each inverted repeat contains core (IRIB) and an auxillary (IRIA) sites [1]. RTP binds to these sequences, resulting in the impediment the replication fork helicase.

RTP Structure

Active RTP is a homodimer composed of 14.5 kDa subunits [8]. The structure of the protein has been determined to a 2.6 A resolution using X-ray crystallography [7]. The RTP protein contains three major structural domains for its specific functionality; DNA-binding, and dimer-dimer interaction domains [7]. The RTP is organized into a dimer by the association of their within the C-terminus [7]. The ‘’ is believed to be involved as the major DNA-binding domain; with the two helices slotting into the major grove and two β strands inserting into the minor grove [7]. This binding interaction is vastly different from the Tus-ter interactions.

RTP Mechanism of Action

An RTP dimer binds the core sequence and the complex formed allows a second dimer to cooperatively to bind to the auxiliary site [5]. In the absence of a core site, the auxiliary site is unable to bind RTP [5]. Furthermore, without the auxiliary site, RTP is unable to block the replication fork, as the interaction of both dimers has been suggested to provide enough DNA binding strength to displace the replication fork [5]. This binding explains how the symmetrical RTP can block replication helicase machinery in an asymmetric manner. The blocking end occurs at the core site, while it is believed that the non-blocking auxiliary site may let replication through as there is less contact points of the dimer to the DNA and the replication machinery coming from this direction is predicted to displace the dimer that is weakly bound to the auxiliary site, which would then displace the dimer bound to the core [5]. Biochemical and mutational studies have identified particular residues that are vital for the functionality of RTP. Mutations within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability [1]. These mutations do not affect dimer-dimer interactions or DNA binding activity and indicate that simple DNA binding is not able to block the replication fork. This provided evidence that RTP and the replication fork machinery interact specifically [1].

The Terminus Utilization Substance (Escherichia coli )

(2ewj).

The E.coli protein that is responsible for termination is a 36kDa protein named Tus (Terminius Utilization Substance) that binds 23bp ter sites and arrests the replication helicase, DnaB, responsible for separating the two strands of DNA [4][9]. Unlike RTP termination sites, the ten E.coli ter sites do not contain inverted sequences or direct repeats and Tus binds as a monomer to a highly conserved core region of 13bp [10]. The tus-ter complex is known to terminate replication by arresting the replication machinery in a in a polar manner however there is great discrepancy in evidence whether Tus specifically interacts or physically blocks the DnaB helicase to arrest its progression.

Tus structure

Tus is a member of the replication termination protein family and has no similarity to any DNA-binding motif that is known, and is organized into two discontinuous domains ( and ) that consist of α helical and β sheets [10]. Two pairs of antiparallel β strands link the N and C terminal protein domains and form the . This provides a large, positivley charged, central cleft into which the double helix (deformed locally) can fit, so that the two α helical domains flank the DNA [10]. The interdomain β strands, which makes up the core region of the 13bp DNA-binding, that sit across from the DNA, accesses a deepened major groove and contacts several of the bases within this groove, and is responsible for ter sequence recognition and binding to DNA [10]. There are 17 sequence-specific interactions between the DNA and the α helixes and β strands presented by Tus, although the majority of these interactions are from the proximinal β sheets [10]. The helicase-blocking or , of Tus consists of α helices and loops from N and C terminal domains [10]. Tus is unrelated structurally to the replication termination protein despite their similar functions [11].

Tus Mechanism of Action

Two models were proposed to explain the mechanism of Tus activity generated by early experiments. The "clamp model" proposed that the Tus-ter complex created a barrier that arrested the progression of the replication machinery from one direction but not the other, by DNA binding [12]. The "interaction model" suggested that a particular region of Tus specifically interacted with the progressing helicase, causing it to halt the fork, and this interaction would only be possible at one face of the protein [4]. The structure of the tus-DNA complex has recently been solved [12]. It suggests that the protein can block helicase approaching from one direction and not the other, without the necessity of specific Tus–helicase interactions. The Tus-Ter complex could act as a physical barrier against the replication fork at the non-permissive face; the α helical regions protrude from the protein around the DNA and block the helicase from accessing the region tightly bound to the DNA [13]. On the other hand, when the helicase advances from the opposite direction it does not encounter the α helical barriers and can disrupt Tus-DNA binding by interrupting the interdomain β strands, causing Tus to be released [13]. This simple model is supported by studies where mutants were screened after exhibiting a reduction in their ability to arrest replication [13]. Most of the mutations occurred in the interdomain β-strands and none of these mutations occurred in the blocking surface that may contact the progressing helicase [13]. However it is important to note that a specific interaction between the blocking face and the helicase cannot be ruled out based on structural studies, and that it if present it may have a role to enhance the physical barrier’s effectiveness.

Interestingly, RTP has been found to arrest replication in E.coli when bound to E. coli specificter sequences. This suggests that the Tus-ter complex provides a physical barrier that is not specific to the replication fork [12]. However there is some evidence to suggest that RTP specifically recognizes the E.coliDnaB helicase allowing it to functionally block replicative progression, and that Tus may act similarly [14]. Mutational analysis a contrahelicase region has shown that mutations within these regions abolish RTP's ability to arrest DnaB. This indicates that protein-protein interactions occur between these two proteins, and further structural analysis has identified that these amino acid region interacts with a hinge region on DnB helicase. These reports mean that specific surfaces of the termination proteins, RTP and Tus, could be recognizing the identical or variable surfaces of the helicases [13].

Biological Significance

The role of the replication fork arrest was believed to be of great importance for the faithful termination of replication, segregation of chromosomes, and inheritance of a stable genome. However recent studies where the rtp and tus genes of B.subtilis and E.coli, respectively, were knocked out, suggested that this role is dispensable [11]. Indeed, bacterial systems that have mutations within these genes can survive in the environment and appear identical in both growth rate and cell morphology compared to wildtype bacteria, suggesting that replication termination is not a requirement for cellular replication [11]. However as it has analogous presence between different bacteria this suggests that this mechanism is beneficial for these prokaryotes. It has recently been suggested that this form of termination may have roles including; aiding the co-ordination and optimization of recombination events preceding replication; preventing over-replication and preventing the harmful affects of clashes that can occur between replication-transcription due to the bacterial bias in gene orientation [11].

As most genes are orientated towards the terminus, from the origin, if replication is not arrested, it progresses into regions being actively transcribed and collides into the transcription RNA polymerase [11]. It is also suggested that termination may occur by specific "dif" sites; conserved sites that are located near the terminus region that are involved in homologous recombination [15]. In fact the dif-terminus hypothesis proposes that termination occurs at or near these sites, where after termination of the replication forks, the two recombinases, XerC and XerD (proteins originating from E.coli), cause site-specific recombination at these dif-sites, and that this would resolve the concatenated chromosomes and complete replication [15]. This mechanism implies that this replication termination by RTP and Tus proteins is merely advantageous to the bacteria and not necessary [15].