User:Bianca Varney/Bacterial Replication Termination

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Revision as of 11:02, 20 May 2011

In most bacterial DNA replication initiation occurs at an origin where, due to the circular nature of the chromosome, the replication forks move bidirectionally to end at approxiametly 180 degrees away, at a specific sequence termini region [1]. Bacterial repliaction termination systems have been well studied in Eschericia coli and Bascillus subtilis. In both systems a trans-acting replication termination protein binds to a specific cis-acting DNA sequences, the replication termini (ter), and the DNA-protein complex arrests the progression of replication forks. The terminator sites are orientated so that protein binding is asymmetric, allowing the complexes to block the replication machinery from only one direction while letting them proceed unimpeded from the other direction [2]. In this way they are said to act in a polar manner. The proteins involved in this termination are non-homologous and differ structurally in E.coli and B.subtilis, although each contains similar contrahelicase activity and performs similar functions in arresting replication.

Termination (Ter) Sites

Replication is terminated in bacterial systems such as E.coli and B.subtilis by a "replication fork trap", studded with termination (Ter) sites which causes the bidirectional forks to pause, encounter and fuse within a region called the terminus region. In E.coli the termination regions are spread across nearly half the chromosome compared to B.subtilis where they cover only ~10%. Termination regions are made up of two groups, opposite to each other, containing inverted sequences for the polar arrest of the replication helicase. In E.coli the 5 Ter sites, J, G, F, B and C are arranged opposed to Ter sites H, I, E, D and A, and can arrest the fork progressing in the clockwise direction and can block the anticlockwise direction, respectively. The replication fork progressing in a clockwise direction will encounter the TerC site first and pause. If the fork progressing from the anticlockwise direction meets the clockwise fork while paused, replication is terminated, however if it does not meet its anti-fork it will proceed until it reaches the next termination site, TerB, where it will pause again, etc [8]. Therefore multiple Ter sites are important as infrequently utilized backups, to ensure that the fork does not leave the terminus region, and that termination is completed. Multiple regions to entrap the replication fork means that if an inactivating mutation arises within a ter site, then arrest can still occur at another ter sequence [6].

Replication Terminator Protein (Bacillus subtilis)

The replication terminator protein (RTP) is a homodimer composed of two 14.5 kDa subunits. The RTP protein contains three major structural domains for its specific functionality, including DNA-binding, DnaB interaction and dimer-dimer interaction domains. Biochemical and mutational studies have identified particular residues that are vital for the functionality of the RT protein. Manna et al have identified that mutation within a hydrophobic region at residues Glu-30 and Tyr-33 causes the loss of contrahelicase ability. 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.

The RTP is organized into a dimer by the association of their long α helices within the C-terminus. The ‘winged helix’ is believed to be involved as the major DNA-binding domain however two of the α helices, at the centre of the protein, and two β strands, in the outer regions, have been suggested to fit adjacently into the major and minor grooves respectively. The unstructured N terminal region is may also have a role in DNA-binding.

Terminus Utilization Substance (Escherichia coli)

The Terminus Utilization Sequence (Tus) structure is organized into two discontinuous domains (N terminal and C terminal) that consist of α helical and β sheets that straddle the DNA helix. Two antiparallel pairs of β strands that form an interdomain providing a large positively charged central cleft, which adopts the double helix. The interdomain β strands, which makes up the DNA-binding domain, accesses a deepened major groove, making base contact. This is responsible for Ter sequence recognition and DNA binding.

X-ray crystallography studies (figure ??) have shown that when bound to Tus the double stranded DNA is extensiblley deformed. The DNA is observed to be significantly unwound, creating a deeper major groove, bending the DNA to approximately 20 degrees and decreasing the helical twist from 34.60 to 29.5,0 caused by the binding of this protein into the DNA.

Tus is unrelated structurally to the replication termination protein despite their similar functions.

Biological Significance

The role of the replication fork arrest was primarily believed to be of great importance for the faithful termination of replication, segregation of chromosomes and faithful 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. 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 cytokinesis [4]. It has recently been suggested that this form of termination may have roles in aiding the co-ordination and optimization of recombination events preceding replication in bacteria, and preventing over-replication. 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. In fact the dif-terminus hypothesis proposes that termination occurs at or near these sites, where after termination of the replication forks, the dif-sites would undergo site-specific recombination, and that this would resolve the dimer chromosomes and complete replication

References