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Nucleic Acids Research, 2000, Vol. 28, No. 9 1893-1898
© 2000 Oxford University Press

DNA strand transfer catalyzed by vaccinia topoisomerase: ligation of DNAs containing a 3' mononucleotide overhang

Chonghui Cheng and Stewart Shuman*

Molecular Biology Program, Sloan-Kettering Institute, 1275 York Avenue, New York, NY 10021, USA

The specificity of vaccinia topoisomerase for transesterification to DNA at the sequence 5'-CCCTT and its versatility in strand transfer have illuminated the recombinogenic properties of type IB topoisomerases and spawned topoisomerase-based strategies for DNA cloning. Here we characterize a pathway of topoisomerase-mediated DNA ligation in which enzyme bound covalently to a CCCTT end with an unpaired +1T nucleotide rapidly and efficiently joins the CCCTT strand to a duplex DNA containing a 3' A overhang. The joining reaction occurs with high efficiency, albeit slowly, to duplex DNAs containing 3' G, T or C overhangs. Strand transfer can be restricted to the correctly paired 3' A overhang by including 0.5 M NaCl in the ligation reaction mixture. The effects of base mismatches and increased ionic strength on the rates of 3' overhang ligation provide a quantitative picture of the relative contributions of +1 T:A base pairing and electrostatic interactions downstream of the scissile phosphate to the productive binding of an unlinked acceptor DNA to the active site. The results clarify the biochemistry underlying topoisomerase-cloning of PCR products with non-templated 3' overhangs.

* To whom correspondence should be addressed. Tel: +1 212 639 7145; Fax: +1 212 717 3623; Email s-shuman@ski.mskcc.org


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Fluorescence Spectroscopy Studies of Vaccinia Type IB DNA Topoisomerase. CLOSING OF THE ENZYME CLAMP IS FASTER THAN DNA CLEAVAGE
J. Biol. Chem., January 4, 2002; 277(1): 345 - 352.
[Abstract] [Full Text] [PDF]


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