TY - JOUR
T1 - Remote Phosphate Contacts Trigger Assembly of the Active Site of DNA Topoisomerase IB
AU - Tian, Ligeng
AU - Claeboe, Christopher D.
AU - Hecht, Sidney M.
AU - Shuman, Stewart
N1 - Funding Information:
This work was supported by NIH grants GM46330 (to S.S.) and CA78415 (to S.M.H.).
PY - 2004/1
Y1 - 2004/1
N2 - Vaccinia topoisomerase IB forms a covalent DNA-(3′-phosphotyrosyl)- enzyme intermediate at its target site 5′-CCCTTp↓ 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.
AB - Vaccinia topoisomerase IB forms a covalent DNA-(3′-phosphotyrosyl)- enzyme intermediate at its target site 5′-CCCTTp↓ 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.
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U2 - 10.1016/j.str.2003.11.025
DO - 10.1016/j.str.2003.11.025
M3 - Article
C2 - 14725763
AN - SCOPUS:1642493666
SN - 0969-2126
VL - 12
SP - 31
EP - 40
JO - Structure
JF - Structure
IS - 1
ER -