From Proteopedia

Revision as of 04:49, 12 May 2011

Replication Termination

Replication is an essential process in all cells. The process copies the chromosomal DNA of the organism to provide the extra copy needed in cell division and is therefore critical in the biological inheritance of genes. In cells with circular chromosomes, replication starts from a single origin proceed with two replication forks moving in opposite directions. This process must be terminated, otherwise it would continue and multiple copies of the chromosome would be made.

The process of terminating replication is performed by replication termination proteins. These proteins bind to specific sequences in the DNA, called Ter sites. This binding provides a physical blockage in the DNA that stops the replication machinery.

In each circular chromosome, there are two sets of Ter sites that appear roughly opposite to the origin of replication. One set blocks the clockwise replication fork while the other traps the anti-clockwise replication fork.

In B. subtilis, the termination protein is called Replication Terminator Protein (RTP), and in E. coli it is Termination Utilisation Substance (Tus).

Interestingly, both these proteins bind to DNA in such a way that they terminate the replication fork in one direction, but simultaneously allow the replication fork to continue in the other direction.

RTP

Structure of RTP

A single RTP monomer consists of four , three and an unstructured domain. The binds to DNA by into the major groove of Ter sites, while the interacts with the minor groove. The N-terminal arm also binds to the Ter site^[1].

Key Features of RTP

Two different RTP B sites have been found to interact with RTP. These are the symmetrical RTP B (sRB) site from TerI (the first Ter site that the clockwise replication fork encounters) and the native RTP B (nRB) site from TerI. These sequences differ only in 6 base pairs – three at the downstream end and three at the upstream end. The downstream changes have no bearing on the structure of RTP since the protein binds the downstream region in both sRB and nRB sequences with similar conformation. Additionally, no base-specific interactions are made in this region. However, the three upstream changes are all located in the major groove of the dsDNA. This is where the α3 helix binds which underlies RTP binding specificity^[2]. Therefore, in binding of RTP monomers to the Ter site, there is a differential binding affinity in A and B sites, due to changes in the nRB site.

This leads into the fact that RTP binds asymmetrically across the nRB site and therefore allows the complex to act as a polar barrier to the replication fork. When the fork approaches the B site (with tight RTP-DNA binding) the fork is unable to progress and is paused. Approaching from the A site does not impede its progress. This polarity can be explained by both the differential binding affinity as explained above and the cooperative binding affect of the RTP monomers. The complex formed between an RTP molecule and the B site facilitates cooperative binding of another RTP monomer to the A site to form a complete RTP-Ter complex^[3].

TUS

Structure of Tus

The Tus protein works as a monomer, each monomer consists of two domains, the amino and carboxy domains. The overall structure of Tus is made up of two separated by central twisted anti-parallel . These structures together form a that accommodates a 13 base pair B-form Ter . This binding involves two β-strands involes contacting the bases and sugar-phosphate back bones from the major groove of the Ter DNA.

References

1. ↑ Pai, S. K., Bussiere, D. E., Wang, F., Hutchinson, C. A., White, S. W. & Bastia, D. (1996) The structure and function of the replication terminator protein of Bacillus subtilis: identification of the ‘winged helix’ DNA-binding domain. EMBO J. 15(12), 3164-3173.
2. ↑ Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. (2007) An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. J. Mol. Biol. 370, 481-491.
3. ↑ Vivian, J. P., Porter, C. J., Wilce, J. A. & Wilce, M. C. J. (2007) An Asymmetric Structure of the Bacillus subtilis Replication Terminator Protein in Complex with DNA. J. Mol. Biol. 370, 481-491.