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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. In most cases, the only cofactor they need for their DNA cleavage reactions is Mg2+. These enzymes thus provide highly amenable systems for analysing the molecular basis 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 DNA. However, by using EcoRV and other restriction enzymes as test systems, we have shown that proteins find their target sites in long DNA chains primarily by multiple dissociation/re-association events within the DNA molecule, and that “sliding” is typically a short range process covering less than 100 bp at a time.

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 below).

DNA looping is a very common event on DNA. It is involved in nearly all genetic processes, including the initiation of DNA replication, transcription and its regulation, genetic recombination and transposition, many pathways for DNA repair. Even so, the restriction enzymes that interact with two copies of their recognition sequence, at separate locations in the DNA, are perhaps the most amenable systems with to study the molecular basis of DNA looping. The complete dissection of a DNA looping reaction (the above pathway) was first obtained by combining the kinetics of DNA cleavage by the SfiI enzyme in bulk solution with single-molecule studies measuring the tethering of a bead by a DNA molecule: the tether gets shorter as the loop is trapped.

Other restriction enzymes trap their loops by different mechanisms. In the case of FokI for example, a monomer of the protein (with one active site) binds to one site on the DNA but it then has to interact with a second molecule on a separate site to give a dimer with two active sites: only then can it cut both DNA strands.

The nature of long-range interactions on DNA, and how they organise themselves to cleave the DNA at two sites, is currently being studied here with several different restriction enzymes, each of which possess special features of interest. For example, BcgI cuts eight bonds at a time – it binds two sites but then makes two double-strand breaks at each site.

Group

Christian Pernstich and Rachel Smith.

Recent publications

Rusling DA, Laurens N, Pernstich C, Wuite GJL, Halford SE. (2012) DNA looping by FokI: the impact of synapse geometry on loop topology at varied site orientations. Nucleic Acids Research. 40: 4977-4987.

Pernstich C, Halford SE. (2012). Illuminating the reaction pathway of the FokI restriction endonuclease by fluorescence resonance energy transfer. Nucleic Acids Research. 40: 1203-1213.

Marshall JJT, Smith RM, Ganguly S, Halford SE. (2011) Concerted action at eight phosphodiester bonds by the BcgI restriction endonuclease. Nucleic Acids Research. 39: 7630–7640.

Sasnauskas G, Zakrys L, Zaremba M, Cosstick R, Gaynor JW, Halford SE, Siksnys V. (2010) A novel mechanism for the scission of double-stranded DNA: BfiI cuts both 3'-5' and 5'-3' strands by rotating a single active site. Nucleic Acids Research. 38: 2399-2410.

View all publications listed on the University of Bristol's publication database