| Summary: | 碩士 === 國立陽明大學 === 生物化學研究所 === 92 === I-HmuI is a homing endonuclease of HNH subfamily, encoded by the group I intron ORF of the DNA polymerase gene in Bacillus subtilis bacteriophage SPO1. It initiates the process of group I intron homing by nicking at the intronless SPO1 DNA polymerase gene allele. I-HmuI bears three motifs: a NUMOD4, an HNH and an IENR1 motif. Among the three motifs, only the HNH motif is functionally and structurally well studied that it contains a beta-beta-alpha-Metal fold and is involved in DNA cleavage. The crystal structure of the nuclease domain of colicin E7 in complex with DNA has shown before the non-specific binding between the HNH motif and DNA. Different from colE7 and colE9, I-HmuI is a site-specific endonuclease which only nicks a double-stranded DNA at a single strand in a specific site. It is not known how I-HmuI selects its site for binding and cleavage.
In order to characterize the function of each motif in I-HmuI, we constructed vectors to over express a full-length and a truncated I-HmuI which only contained the C-terminal IENR1 motif. We found that the full-length I-HmuI had optimal endonuclease activity at ~45 degree C in the presence of divalent metal ions, Mg2+ or Mn2+. The Ca2+ ions, as well as Na+ ions, inhibited the enzyme activity. The truncated IENR1 motif was only capable to bind DNA, however much weaker than that of the wild-type enzyme, and it cannot nick the DNA substrate. These results indicate that IENR1 motif is likely a site-specific DNA-binding motif which helps I-HmuI to recognize its specific target for the cleavage carried out by the non-specific endonuclease HNH motif. Co-crystallization of full-length and truncated I-HmuI with DNA only gave precipitate and tiny crystalline-like material. Improvement of the protein purification process and the crystallization conditions will be carried out eventually for the determination of I-HmuI/DNA structure by X-ray diffraction methods.
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