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KMID : 0578319940040050081
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Molecules and Cells
1994 Volume.4 No. 5 p.81 ~ p.86
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DNA Sequence-dependent Cleavage Sites of Restriction Endonuclease HphI
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Cho Seo-Hee
kang Chang-Won
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Abstract
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A class IIS restriction endonuclease 17phI has been known to make a staggered cut always at the 8th base pair toward the downstream of its recognition sequence 5´GGTGA3´, producing one-base 3´ protruding ends, regardless of the DNA sequence around the cleavage site. Since the cleavage sites of HphI are separate from its recognition sites, the effects of mutations around a cleavage site have been studied using a phage SP6 promoter-containing plasmid pSP64 which has a HphI cleavage site near the transcription initiation site. The exact cutting site of the lower strand was determined to be after the 8th nucleotide downstream of the recognition site, indicating a staggered cut at the 9th base pair down-stream. In addition, the same cutting site in the mutants carrying a few base pair deletions around the cleavage site is shown to be shifted. These variations were not affected by any changes in reaction conditions. These results suggest that the HphI cleavage site shifts depending on the DNA sequence, which might affect the DNA helical structure.
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