DNA contacts by protein domains of the molluscum contagiosum virus type-1B topoisomerase.
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.
Hwang
Y
Y
Infectious Disease Laboratory, The Salk Institute for Biological Studies, La Jolla, California, 92037, USA.
Park
M
M
Fischer
W H
WH
Burgin
A
A
Bushman
F
F
D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D017124:Conserved Sequence / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004278:DNA, Superhelical / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D008977:Molluscum contagiosum virus / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D009946:Organothiophosphorus Compounds / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D012965:Sodium Chloride / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D004278:DNA, Superhelical; D009946:Organothiophosphorus Compounds; D010446:Peptide Fragments; D011994:Recombinant Proteins; D015636:Magnesium Chloride; D012965:Sodium Chloride; D004247:DNA; D014357:Trypsin; D004264:DNA Topoisomerases, Type I