DNA contacts by protein domains of the molluscum contagiosum virus type-1Btopoisomerase

All poxviruses studied encode a type 1B topoisomerase that introduces trans ient nicks into DNA and thereby relaxes DNA supercoils. Here we present a s tudy of the protein domains of the topoisomerase of the poxvirus molluscum contagiosum (MCV), which allows us to specify DNA contacts made by differen t domains. Partial proteolysis of the enzyme revealed two stable domains se parated by a protease-sensitive linker. A fragment encoding the linker and carboxyl-terminal domain (residues 82-323) was overexpressed in Escherichia coil and purified. MCV topoisomerase (MCV-TOP)(82-323) could relax superco iled 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 sequenc e-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 impo rtant in the initial cleavage step. MCV-TOP(82-323) differed from the full- length protein in showing reduced sensitivity to mutations at a position wi thin the 5'-(T/C)CCTT-3' recognition site, consistent with a model in which the amino-terminal domain contacts this region. These findings provide ins ight into the division of labor within the MCV-TOP enzyme. (C) 1999 Academi c Press.