All poxviruses studied encode a type 1B topoisomerase that introduces transient 
nicks into DNA and thereby relaxes DNA supercoils. Here we present a study of 
the protein domains of the topoisomerase of the poxvirus molluscum contagiosum 
(MCV), which allows us to specify DNA contacts made by different domains. 
Partial proteolysis of the enzyme revealed two stable domains separated by a 
protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal 
domain (residues 82-323) was overexpressed in Escherichia coli and purified. MCV 
topoisomerase (MCV-TOP)(82-323) could relax supercoiled plasmids in vitro, 
albeit with a slower rate than the wild-type enzyme. MCV-TOP(82-323) was 
sensitive to sequences in the favored 5'-(T/C)CCTT-3' recognition site and also 
flanking DNA, indicating that some of the sequence-specific contacts are made by 
residues 82-323. Assays of initial binding and covalent catalysis by 
MCV-TOP(82-323) identified the contacts flanking the 5'-CCCTT-3' sequence at 
+10, +9, -2, and -3 to be important. Tests with substrates containing a 
5-bridging phosphorothiolate that trap the cleaved complex revealed that correct 
contacts to the flanking sequences were important in the initial cleavage step. 
MCV-TOP(82-323) differed from the full-length protein in showing reduced 
sensitivity to mutations at a position within the 5'-(T/C)CCTT-3' recognition 
site, consistent with a model in which the amino-terminal domain contacts this 
region. These findings provide insight into the division of labor within the 
MCV-TOP enzyme.