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Nucleic Acids Research, Vol 24, Issue 21 4171-4175, Copyright © 1996 by Oxford University Press

ARTICLES

Effects on NaeI-DNA recognition of the leucine to lysine substitution that transforms restriction endonuclease NaeI to a topoisomerase: a model for restriction endonuclease evolution

K Jo and MD Topal
Lineberger Comprehensive Cancer Center and Department of Pathology, University of North Carolina Medical School, Chapel Hill 27599-7295, USA.

Substituting lysine for leucine at position 43 (L43K) transforms NaeI from restriction endonuclease to topoisomerase and makes NaeI hypersensitive to intercalative anticancer drugs. Here we investigated DNA recognition by Nael-L43K. Using DNA competition and gel retardation assays, NaeI-L43K showed reduced affinity for DNA substrate and the ability to bind both single- and double-stranded DNA with a definite preference for the former. Sedimentation studies showed that under native conditions NaeI-L43K, like NaeI, is a dimer. Introduction of mismatched bases into double-stranded DNA significantly increased that DNA's ability to inhibit NaeI-L43K. Wild-type NaeI showed no detectable binding of either single-stranded DNA or mismatched DNA over the concentration range studied. These results demonstrate that the L43K substitution caused a significant change in recognition specificity by NaeI and imply that NaeI-L43K's topoisomerase activity is related to its ability to bind single-stranded and distorted regions in DNA. A mechanism is proposed for the evolution of the NaeI restriction- modification system from a topoisomerase/ligase by a mutation that abolished religation activity and provided a needed change in DNA recognition.
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