Evolution of DNA restriction endonucleases and the creation of new endonucleases for DNA manipulation

  • Dryden, David (Principal Investigator)

Project Details

Description

Restriction endonucleases have been the foundation of molecular biology since the 70's. Even today when sophisticated
designer nucleases such as site-specific Zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) can be created
for gene targeting and restriction enzymes are considered to be simple tools, restriction enzymes are still used at some
stage in almost every molecular biology/biotechnology project for DNA manipulation. New restriction enzymes are still
being sought to expand the repertoire of DNA targets and new uses discovered.
The Type I restriction enzymes cut DNA on either side of their target (invariably a bipartite sequence such as
AACN6GTGC) but at variable distances (usually thousands of bp distant). The random cutting sites preclude commercial
use of these enzymes. The Type IIB cut at defined short distances on either side of their bipartite target (e.g. BcgI cuts
|10/12|CGAN6TGC|12/10|) and this specificity makes them commercially valuable.
We have recently determined the first structures of the Type I restriction enzymes. These show how domain deletion and
fusion can be achieved in three-D space to create Type IIB (and IIG) restriction enzymes from Type I restriction enzymes.
The number of Type IIB/IIG restriction enzymes known is currently small (1000).
Thus the question we wish to ask is whether hundreds of Type I restriction enzymes can be easily converted into
commercially viable Type IIB/IIG enzymes with new target specificities?
The Type I and IIB enzymes additionally prefer DNA substrates with two copies of the target sequence, thus the proposed
new enzymes should demonstrate activity on DNA containing adjacent copies of the target in much the same manner as
the ZFNs and TALENs. Therefore they may be functional in gene targeting as well as genetic engineering.

Layman's description

Genetic engineering, essential to the progress of all current biological research, would be impossible without a class of
catalysts termed restriction enzymes. These allow the cutting and pasting of segments of DNA to test hypotheses and to
create novel routes to vital healthcare products such as insulin. Arguably, it is fair to say that nothing in modern biology,
biotechnology and biomedical research would be possible without them. The number of restriction enzymes known is large
and many have been commercialised. However new enzymes are still being sought and new uses for them are still being
invented. We have recently solved the structure of the first restriction enzyme to be discovered and purified in 1968. These
"Type I" restriction enzymes have not so far had impact on healthcare, research or wealth creation despite their abundance
in natural populations of bacteria and their impact on slowing down the transfer of DNA encoding, for example, antibiotic
resistance and the creation of "superbugs". However our new structure shows how Type I restriction enzymes can be
converted into valuable products for research and healthcare. Since thousands of Type I restriction enzymes are known to
exist, this ability to convert them promises a veritable bonanza of new and useful restriction enzymes. We propose to
perform this conversion process on the Type I restriction enzymes. As well as aiding new research these new enzymes
may well prove valuable new tools for gene targeting and editing; a relatively new area studying the genetics of complex
organisms particularly with an emphasis on understanding and eventually treating human genetic diseases.
StatusFinished
Effective start/end date1/01/1330/04/16

Funding

  • BBSRC: £738,121.00

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