Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking.
Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and cleaves duplex DNA at sites containing the sequence 5'-(T/C)CCTT. We report the identification of Tyr70 as the site of contact between the enzyme and the +4C base of its target site. This was accomplished by UV-crosslinking topoisomerase to bromocytosine-substituted DNA, followed by isolation and sequencing of peptide-DNA photoadducts. A model for the topoisomerase-DNA interface is proposed, based on the crystal structure of a 9 kDa N-terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C base and Tyr72 is situated near the +3C base. Mutational analysis indicates that Tyr70 and Tyr72 contribute to site recognition during covalent catalysis. We propose, based on this and other studies of the vaccinia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well-conserved 20 kDa C-terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N-terminal domain, which is less conserved between viral and cellular proteins. Division of function among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.
Sekiguchi
J
J
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Shuman
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism* / Q000528:radiation effects; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D004951:Esterification; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D014443:Tyrosine / Q000378:metabolism; D014616:Vaccinia virus / Q000201:enzymology
D004268:DNA-Binding Proteins; D014443:Tyrosine; D004247:DNA; D004264:DNA Topoisomerases, Type I