Chemical and traditional mutagenesis of vaccinia DNA topoisomerase provides insights to cleavage site recognition and transesterification chemistry

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 Tyr²⁷⁴ 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-OCH₃ 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 ⁺⁵C⁺⁴C⁺³T⁺²T⁺¹p ↓ N (where ↓ denotes the cleavage site). A signature α-helix of the poxvirus TopIB (¹³²GKMKYLKENETVG¹⁴⁴) 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 Tyr¹³⁶ highlight the importance of van der Waals interactions with the 3′-G⁺⁴pG⁺³p dinucleotide of the nonscissile strand for DNA cleavage and supercoil relaxation. Asn¹⁴⁰ and Thr ¹⁴² donate hydrogen bonds to the pro-(S_p)-oxygen of the G⁺³pA⁺² phosphodiester of the nonscissile strand. Lys ¹³³ and Lys¹³⁵ 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.