Abstract
We studied the interaction between topoisomerase I and a nicked DNA substrate to determine how the nick permits Escherichia coli topoisomerase I to catenate and knot duplex DNA rings. The presence of just a single nick in a 6600-base pair DNA increased the amount of DNA bound to topoisomerase I by 6-fold. The enzyme acts at the nick, as shown by linearization of nicked circles and covalent attachment of an enzyme molecule opposite the nick. DNA breaks are also introduced by the enzyme at sites not opposite to a nick, but three orders of magnitude less efficiently. The break induced by the enzyme is within several base pairs of the nick and on the complementary strand, but the exact site cut is dictated by DNA sequence requirements. Because these sequence requirements are identical to those for cutting of single-stranded DNA, we conclude that the enzyme stabilizes a denatured region at the nick. Breaks in single-stranded DNA occur 98% of the time when a C residue is four bases to the 5' side unless G is adjacent and 5' to the break. For a DNA circle nicked at a unique location, the efficiency of DNA breakage opposite the nick correlates with the rate of catenation. We present a unified model for the relaxation, catenation, and knotting reactions of topoisomerase I in which the enzyme induces a break in a single-stranded region, but bridges that break with covalent and noncovalent interactions and allows passage of one duplex or single-stranded DNA segment.
Highlights
From the $Departmentof Molecular Biology, University of California, Berkeley, California94720 and the §Departmentof Biochemistry, University of Chicago, Chicago, Illinois 60637
Since the relaxation rate depends on superhelical density, this result implies that the cleavage product is structurallyanalogous to a reaction intermediate
Previous studies have shown that E. coli topoisomerase I can catenate and knoctircles of nicked, duplex DNA [10,11,12]
Summary
From the $Departmentof Molecular Biology, University of California, Berkeley, California94720 and the §Departmentof Biochemistry, University of Chicago, Chicago, Illinois 60637. Gaps of limited size were created by treating 5'-32P-labeled substrate DNA nicked at the Am11 site (Fig. 6) with E. coli exonuclease I11under conditions that result in theremoval of 8-20 bases/min from the 3' end [19].
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