A MAMMALIAN SITE-SPECIFIC ENDONUCLEASE
MCKENNA, WILLIAM GILLIES
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Recent evidence suggests that site-specific recombination may be important both for genetic rearrangement on an evolutionary scale and for gene control during differentiation. A consideration of the mechanism of recombination suggested that a site specific endonuclease might function to initiate such an event. A search was undertaken for such an activity in mammalian cells using as a model the bacterial restriction enzymes.;An activity has been identified from a variety of mammalian sources which cleaves native viral and repetitive DNA to yield apparently specific fragments when analysed by gel electrophoresis. The activity has been purified 750 fold by ionexchange chromatography. It has a pH optimum of 7.5, an absolute requirement for magnesium and is inhibited by high salt concentrations. By glycerol gradient analysis it has a sedimentation coefficient of 4.5s and may exist as a dimer of 6.4s corresponding to molecular weights of 65000 and 130,000.;The similarities between this activity and the bacterial restriction enzymes are very superficial. The specific DNA fragments produced by the enzyme are intermediate products in the degradation of DNA rather than end products. The activity will degrade both single and double stranded DNA though it shows site specificity only on double stranded substrates. DNA is degraded without releasing acid soluble nucleotides and the final end products are about 100 bp long.;The activity has been used to explore the structure of a variety of mammalian repetitive DNAs. Some repetitive DNAs, such as African green monkey component DNA, contain sites for which the nuclease has high affinity and are degraded very specifically. Others, such as calf satellite 1 DNA, are degraded apparently randomly by the nuclease. Some species, such as the guinea pig contain repetitive components of both types. In most cases the repeat length seen by the homologous endonuclease is identical to that identified by bacterial restriction enzymes but in one case, mouse satellite DNA, it is not. The significance of these observations is discussed.;Finally, using a completely sequenced repetitive DNA, the preferred site of cleavage of the mammalian endonuclease has been deduced.
- Theses and Dissertations