| Summary: | 碩士 === 輔仁大學 === 生物學研究所 === 83 === The known gene products of IS2 include InsA and InsAB'. This
study is aimed to determine the functional domain of InsA and
InsAB' for further understanding of the transposition mechanism
of IS2. Result showed that InsA protein formed a dimer.
Deletion of the 15 residues at its C-terminus but not the 10
residues at the N-terminus abolished the capability of the
protein to form a dimer, indicating that the protein-protein
interaction domain is located at the C-terminus of InsA. To
determine the DNA binding domain of InsA, the gel retardation
assay was performed. Result revealed that deletion of the 15
amino-acid residues at its C-terminus has no effect on the DNA
binding ability of InsA. These facts indicated that the DNA
binding domain of InsA is located at its N-terminus. However,
the peptide containing the InsA N-terminal 60 residues is not
sufficient for the DNA binding. A previous study demonstrated
that InsAB' is the transposase of IS2. The N-terminal 106 amino-
acid residues of InsA and InsAB' are identical. However,
besides P site InsAB' also binds the inner ends of RIR and LIR
in IS2. This suggested that the DNA binding domain of InsAB'
may contain different regions: one for P site and the other for
LIR and RIR. InsAB'DM was found to bind the RIR and LIR of IS2
with decreased binding affinity. This finding suggests that the
LIR and RIR binding domain of InsAB' is in the proximity of the
mutation site made in InsAB'DM. Transposase should have non-
specific DNA binding activity. The possibility was confirmed by
that InsAB'indeed bound the P1 phage DNA fragment that contains
the hot spot for IS2 transposition. However, the non-specific
DNA binding domain of InsAB' remains to be investigated.
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