User:David McDonald/Replication Termination Protein
From Proteopedia
As with most bacteria, DNA replication of the circular chromosome of B. Subtilis occurs in a bi-directional fashion, starting from a common origin of replication (ori) and ending in the termination region, approximately 180o from the ori. The two replication forks are forced to meet in the termination region by replication termination proteins (RTPs) complexed to specific, unidirectional DNA termination sits (Ter sequences) termination region and arrest the action of the replication fork in a directional manner. That is, there are RTP:Ter complexes which stop replication in the clockwise and in the anti-clockwise direction. The clockwise replication fork is unaffected by the RTP:Ter complexes for the anti-clockwise fork and vice versa.
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DNA Replication in Bacteria
Structure
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contains 122 amino acid residues and is an example of a winged helix structure, in the α+β protein folding family, containing four α-helices and two β-strands[1]. The structure is named a "winged helix" due to the central , flanked by comprised of the two β-strands and the loops between them.
In a cell, RTP exists as a homodimer, where the monomer subunits are tightly associated through antiparallel coiled-coil interactions between the α4 helices to form the functional . This dimer is held together by between the two α4 helices.
For crystallographic studies, a mutant of RTP (C110S) was created to avoid possible unwanted disulfide bond formation. In this mutant, the Cysteine at position was changed to a Serine residue. It was shown that this did not result in a change in solution dimerisation or DNA binding functionality[2].
DNA Binding
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In order to terminate chromosomal replication, RTP must complex with the DNA. Two dimers of RTP bind at the Ter site, a 37bp region of DNA containing two pseudosymmetric overlapping 21 base pair half-sites A and B.[1] The B site displays significantly higher affinity for the RTP dimer[2], however it has been shown that it is necessary for both A and B to be occupied in order for replication fork termination to occur.[2]
The formation mechanism and subsequent structure this RTP-DNA complex has been a topic of some debate. Previously, it was thought that the complex was [3], with mediated by the α3 helix as predicted, but also the α1 helix and, surprisingly, the α1-α2 loop region. More specifically, the binding was attributed to Arg59, His54, Thr55 and Tyr58 of α3; Tyr33, Gly34 and Leu35 of α 1- α2 loop region and Gln15 of α1 [3]. This structure was obtained with a symmetric B site analogue known as sRB as opposed to the wild type Ter sequence, which differs by only 6 bases.
The observed symmetry was in contrast to the known polarity of the RTP-Ter site. Further research using the wild-type Ter sequence (nRB) revealed a distinct in both the RTP dimer and the DNA[4]. Although the interacting sequences remained the same, the downstream monomer displayed a confirmation, where the β2-loop-β3 structure makes contact with the phosphate backbone. Conversely, the upstream monomer displays a confirmation, with the wing making contact with the upstream minor groove.
Replication Termination
Works Cited
1. Bussiere, DE, Bastia, D and White, SW. (1995)Crystal structure of replication terminator protein of B. subtilis at 2.6 Å. Cell. 80: 651-660.
2. J.P. Vivian, A.F. Hastings, I.G. Duggin, R.G. Wake, M.C.J. Wilce, and J.A. Wilce (2003) “The impact of single cysteine residue mutations on the replication terminator protein” Biochem. Biophys. Res. Commun. 310(4):1096-
