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The role of metals in catalysis by the restriction endonuclease Bam HI

Abstract

Type II restriction enzymes are characterized by their remarkable specificity and simplicity. They require only divalent metals (such as Mg2+ or Mn2+) as cofactors to catalyze the hydrolysis of DNA. However, most of the structural work on endonucleases has been performed in the absence of metals, leaving unanswered questions about their mechanisms of DNA cleavage. Here we report structures of the endonuclease BamHI–DNA complex, determined in the presence of Mn2+ and Ca2+, that describe the enzyme at different stages of catalysis. Overall, the results support a two-metal mechanism of DNA cleavage for BamHI which is distinct from that of EcoRV.

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Figure 1: BamHI active site.
Figure 2: A stereo view of the refined 2Fo - Fc map around the R active site of the calcium bound pre-reactive BamHI–DNA complex.
Figure 3: A stereo view of the refined 2Fo - Fc map around the R active site of the manganese bound post-reactive BamHI–DNA complex.
Figure 4: a, A comparison of the pre-reactive (blue) and the post-reactive (red) BamHI–DNA complexes.
Figure 5: A comparison of the BamHI (blue) and the EcoRV (magenta) active sites, prior to DNA cleavage.

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Acknowledgements

We thank I. Schildkraut for stimulating discussions; R. Kucera for the purified protein; the staff at CHESS and C. Escalante, J. Hirsch, E. Jacobson, and D. Wah for help with data collection; G. Mogilnitsky and L. Shen for technical help;

I. Schildkraut, L. Shapiro, and D. Wah for comments on the manuscript. This work was supported by a grant from the National Institutes of Health (A.K.A.) H.V. was supported by a Fulbright/CONACYT scholarship.

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Viadiu, H., Aggarwal, A. The role of metals in catalysis by the restriction endonuclease Bam HI. Nat Struct Mol Biol 5, 910–916 (1998). https://doi.org/10.1038/2352

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