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[摘要]：SgrAI is a type II restriction endonuclease that cuts an unusually long recognition sequence and exhibits allosteric self-modulation of DNA activity and sequence specificity. Precleaved primary site DNA has been shown to be an allosteric effector [Hingorani-Varma, K., and Bitinaite, J. (2003) J. Biol. Chem. 278, 40392-40399], stimulating cleavage of both primary (CR vertical bar CCGGYG, where the vertical bar indicates a cut site, R denotes A or G, and Y denotes C or T) and secondary [CR vertical bar CCGGY(A/C/T) and CR vertical bar CCGGGG] site DNA sequences. The fact that DNA is the allosteric effector of this endonuclease suggests at least two DNA binding sites on the functional SgrAI molecule, yet crystal structures of SgrAI [Dunten, P. W., et al. (2008) Nucleic Acids Res. 36, 5405-5416] show only one DNA duplex bound to one dimer of SgrAI. We show that SgrAI forms species larger than dimers or tetramers [high-molecular weight species (HMWS)] in the presence of sufficient concentrations of SgrAI and its primary site DNA sequence that are dependent on the concentration of the DNA-bound SgrAI dimer. Analytical ultracentrifugation indicates that the HMWS is heterogeneous, has sedimentation coefficients of 15-20 s, and is composed of possibly 4-12 DNA-bound SgrAI dimers. SgrAI bound to secondary site DNA will not form HMWS itself but can bind to HMWS formed with primary site DNA and SgrAI. Uncleaved, as well as precleaved, primary site DNA is capable of stimulating HMWS formation. Stimulation of DNA cleavage by SgrAI, at primary as well as secondary sites, is also dependent on the concentration of primary site DNA (cleaved or uncleaved) bound SgrAI dimers. SgrAI bound to secondary site DNA does not have significant stimulatory activity. We propose that the oligomers of DNA-bound SgrAI (i.e., HMWS) are the activated, or activatable, forms of the enzyme. |
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