User:Bianca Varney/Bacterial Replication Termination

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Revision as of 11:43, 19 May 2011

Bacterial replication termination is mediated by replication terminator proteins that bind to polar inverted repeats approximately 180 degrees from the origin of replication (OriC). Replication terminator proteins bind bacterial DNA at termination (Ter) sites. When the replication forks meet with terminator proteins bound to Ter sites, replication is arrested, and DNA polymerase falls off the bacterial chromosome. However, protein-Ter interactions are orientation specific, and will only arrest the replication forks traveling in on one direction; either clockwise or anticlockwise. This means that one replication fork will be arrested at a Ter site, while the other fork, traveling in the opposite direction, will pass through the site unimpeded, allowing the entire bacterial chromosome to be copied. The area loaded with Ter sites in the chromosome is called a "replication fork trap".

Image:Tersitessmall.png

Mechanism

Bacterial replication termination has been well studied in E. coli and B. subtilis. The proteins involved in this termination process differ structurally in these two bacterium, although each contains similar contrahelicase activity and performs similar functions in arresting replication. The replication termini sequences are located in two clusters approximately 180 degrees from the origin of replication, and are orientated such that each cluster has opposite polarity and are therefore inverted repeats. This means that counterclockwise replication forks can move beyond the first cluster of ter sites, the left terminus, but are arrested at the second cluster, the right terminus, containing correct polarity. Similarly, the clockwise fork will proceed through the left cluster but will be arrested at the right. The bipartite Ter nucleotide sequence is overlapping and each inverted repeat contains a core (IRIB) and an axillary (IRIA) sites. RTP binds to these sequences, resulting in the impediment the replication fork helicase.

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