Remote phosphate contacts trigger assembly of the active site of DNA topoisomerase IB.
Vaccinia topoisomerase IB forms a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate at its target site 5'-CCCTTp downward arrow in duplex DNA. The contributions of backbone electrostatics and individual phosphate oxygens to the transesterification reaction were probed by introducing 22 single Rp and Sp methylphosphonate diastereomers at 11 positions flanking the cleavage site. Methyl groups at eight positions (four on the scissile strand and four on the nonscissile strand) inhibited the rate of single-turnover cleavage by factors of 50-50,000. Stereospecific interference was observed at several phosphates, thereby distinguishing simple electrostatic contributions from putative specific polar contacts to either the pro-Sp or pro-Rp oxygens. The functionally relevant phosphate oxygens are located on the minor groove face of the helix on which the scissile phosphodiester resides. Our findings, combined with available crystal structures of vaccinia and human topoisomerase IB, show how specific phosphate contacts remote from where chemistry occurs are critical for assembly of the active site.
Tian
Ligeng
L
Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10021, USA.
Claeboe
Christopher D
CD
Hecht
Sidney M
SM
Shuman
Stewart
S
D001483:Base Sequence; D001665:Binding Sites; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009711:Nucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D014615:Vaccinia
D009711:Nucleotides; D009943:Organophosphorus Compounds; D010710:Phosphates; C032627:methylphosphonic acid; D004264:DNA Topoisomerases, Type I