Abstract
We present a mutational analysis of vaccinia topoisomerase that highlights the contributions of five residues in the catalytic domain (Phe-88 and Phe-101 in helix alpha1, Ser-204 in alpha5, and Lys-220 and Asn-228 in alpha6) to the DNA binding and transesterification steps. When augmented by structural information from exemplary type IB topoisomerases and tyrosine recombinases in different functional states, the results suggest how closure of the protein clamp around duplex DNA and assembly of a functional active site might be orchestrated by internal conformational changes in the catalytic domain. Lys-220 is a constituent of the active site, and a positive charge at this position is required for optimal DNA cleavage. Ser-204 and Asn-228 appear not to be directly involved in reaction chemistry at the scissile phosphodiester. We propose that (i) Asn-228 recruits the Tyr-274 nucleophile to the active site by forming a hydrogen bond to the main chain of the tyrosine-containing alpha8 helix and that (ii) contacts between Ser-204 and the DNA backbone upstream of the cleavage site trigger a separate conformational change required for active site assembly. Mutations of Phe-88 and Phe-101 affect DNA binding, most likely at the clamp closure step, which we posit to entail a distortion of helix alpha1.
Highlights
The 314-amino acid vaccinia virus topoisomerase consists of two structural modules
When augmented by structural information from exemplary type IB topoisomerases and tyrosine recombinases in different functional states, the results suggest how closure of the protein clamp around duplex DNA and assembly of a functional active site might be orchestrated by internal conformational changes in the catalytic domain
The poxvirus topoisomerase has not been crystallized in the DNA-bound state, extensive biochemical data (6 –9) plus the structure of the human topoisomerase I-DNA cocrystal [10] support a model of the vaccinia topoisomerase-DNA complex [3] whereby the N-terminal domain interacts with the major groove on the face of the helix opposite the cleavage site while the C-terminal catalytic domain interacts with the minor groove on the face of the helix that contains the scissile phosphodiester
Summary
The 314-amino acid vaccinia virus topoisomerase consists of two structural modules (see Fig. 1). The extent of cleavage of 0.3 pmol of DNA substrate by the wild-type topoisomerase during a 5-min reaction was proportional to added enzyme and reached saturation at 37 ng of protein at which point Ͼ90% of the input CCCTT DNA strand became covalently bound.
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