title: Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry.
abstract: Vaccinia DNA topoisomerase IB (TopIB) relaxes supercoils by forming and resealing a covalent DNA-(3'-phosphotyrosyl)-enzyme intermediate. Here we gained new insights to the TopIB mechanism through "chemical mutagenesis." Meta-substituted analogs of Tyr(274) were introduced by in vitro translation in the presence of a chemically misacylated tRNA. We report that a meta-OH reduced the rate of DNA cleavage 130-fold without affecting the rate of religation. By contrast, meta-OCH(3) and NO(2) groups elicited only a 6-fold decrement in cleavage rate. We propose that the meta-OH uniquely suppresses deprotonation of the para-OH nucleophile during the cleavage step. Assembly of the vaccinia TopIB active site is triggered by protein contacts with a specific DNA sequence 5'-C(+5)C(+4)C(+3)T(+2)T(+1)p downward arrowN (where downward arrow denotes the cleavage site). A signature alpha-helix of the poxvirus TopIB ((132)GKMKYLKENETVG(144)) engages the target site in the major groove and thereby recruits catalytic residue Arg(130) to the active site. The effects of 11 missense mutations at Tyr(136) highlight the importance of van der Waals interactions with the 3'-G(+4)pG(+3)p dinucleotide of the nonscissile strand for DNA cleavage and supercoil relaxation. Asn(140) and Thr(142) donate hydrogen bonds to the pro-(S(p))-oxygen of the G(+3)pA(+2) phosphodiester of the nonscissile strand. Lys(133) and Lys(135) interact with purine nucleobases in the major groove. Whereas none of these side chains is essential per se, an N140A/T142A double mutation reduces the rate of supercoil relaxation and DNA cleavage by 120- and 30-fold, respectively, and a K133A/K135A double mutation slows relaxation and cleavage by 120- and 35-fold, respectively. These results underscore functional redundancy at the TopIB-DNA interface.
authors: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10065, USA.
authors: Lyudmila
authors: L
authors: Yakovleva
authors: Shengxi
authors: S
authors: Chen
authors: Sidney M
authors: SM
authors: Hecht
authors: Stewart
authors: S
authors: Shuman
mesh_terms: D019943:Amino Acid Substitution; D001665:Binding Sites / Q000502:physiology; D004264:DNA Topoisomerases, Type I / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D017433:Protein Structure, Secondary / Q000502:physiology; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D014764:Viral Proteins; D004264:DNA Topoisomerases, Type I