Research groups

Research

Long range interactions by restriction enzymes on DNA.

Essentially all of the biological functions of DNA depend on proteins that interact with DNA. The Type II restriction enzymes recognise specific DNA sequences and cleave the DNA at fixed locations at or near their recognition sites. They have relatively simple subunit structures, typically dimers of identical subunits with each subunit cleaving one strand of the DNA, though some exist as monomers and others as tetramers. The only cofactor they need for their DNA cleavage reactions is Mg2+. These enzymes thus provide highly amenable systems for analysing the molecular mechanisms of DNA-protein interactions. Before a protein can interact with a specific site in a long DNA, it first has to find that site amid a very large number of non-cognate sites. The initial binding of a protein to the DNA is most unlikely to be at the specific site and will instead be anywhere along DNA. The protein then has to translocate from the initial site to the specific site.

 

It had been widely thought that the translocation occurs by the protein “sliding” along the length of the DNA (see Figure). However, recent work in this laboratory has shown that the EcoRV endonuclease finds its target site by “jumping” rather than “sliding”: i.e., by multiple dissociation/re-association events within a single molecule of DNA.

Further studies on target-site location by other restriction enzymes are currently under way here. EcoRV is a dimeric protein that cleaves each recognition site in a separate reaction. However, most restriction enzymes have to interact with two copies of their target sites before they can cleave DNA. For example, SfiI is a tetramer that binds first to one recognition site, via two of its subunits, and then to a second site, via the other two subunits, thus holding the DNA between the sites in a loop (see Figure).

In other cases, one molecule of the restriction enzyme binds to one site and a second molecule to a separate site, but DNA cleavage occurs only after the two DNA-bound enzymes interact with each other, to trap the intervening DNA in a loop. Long-range communications between distant DNA sites play crucial roles in almost all genetic events. The restriction enzymes that interact with two DNA sites thus illustrate a universal process in DNA transactions. They allow for the molecular analysis of the protein-DNA and the protein-protein interactions that underpin this process.

The nature of action at a distance on DNA is currently being studied here with SfiI and also with several other restriction enzymes that bridge two DNA sites by different mechanisms: SgrAI, FokI and BspMI.

Group

David Rusling, Kelly Sanders, Yana Kovacheva, Alistair Jacklin, Christian Pernstich, Susan Retter, Jacqui Marshall and Fiona Preece.

Recent publications

Sasnauskas, G., Connolly, B. A., Halford, S. E. & Siksnys, V. (2008). Template-directed addition of nucleosides to DNA by the BfiI restriction enzyme. Nucleic Acids Res. 36, 3969-3977.

Bellamy, S. R. W., Mina, P., Retter, S. E & Halford, S. E. (2008). Fidelity of DNA sequence recognition by the SfiI restriction endonuclease is determined by communications between its two DNA-binding sites. J. Mol. Biol. 384, 557-563.

Halford, S. E. (2009). An end to 40 years of mistakes in DNA-protein association kinetics? Biochem. Soc. Trans., 37, 343-348.

Sanders, K. L., Catto, L. E., Bellamy, S. R. W. &. Halford, S. E. (2009). Targeting individual subunits of the FokI restriction endonuclease to specific DNA strands. Nucleic Acids Res, in press.