Diverse energetic effects of charge reversal mutations of poxvirus topoisomerase IB.

摘要

The topological state of the genome is crucial to its functionality. Many cellular functions alter the genomic topology. Reactions that introduce positive supercoils create torsional stress that can prevent protein binding. On the other hand, negative supercoils decrease the duplex melting temperature. In order to maintain a functional genome, the cell uses topoisomerases to introduce or remove supercoils as necessary. A key aspect of the reaction mechanism of type IB topoisomerases is the controlled unwinding of DNA supercoils while the enzyme is transiently bound to one strand of the DNA duplex via a phosphotyrosyl linkage. In this complex, the mobile segment of the bound DNA, downstream from the cleavage site, must rotate around the helical axis, requiring that interactions with the enzyme must break and reform multiple times during the course of supercoils unwinding. A crystal structure of variola topoisomerase Ib (vTopo) bound to DNA shows several positively charged side chains that interact with the downstream mobile and upstream rigid segments, suggesting that these groups may play a role in catalysis, including the processive unwinding of supercoils. We have mutated three such residues, R67, K35 and K271, to Ala and Glu and determined the energetic effects of these mutations at each point along the reaction coordinate of vTopo. R67 interacts with a phosphate group in the rigid DNA segment across from the site of DNA strand cleavage. The ∼30-fold damaging effects of the R67A and R67E mutations were primarily on the phosphoryl transfer step. Removal of the K35 interaction shows similar mutational effects as R67, even though this residue interacts with the mobile segment three base pairs away from the cleavage site. The K271 mutations, which interact with the mobile region even further from the site of covalent linkage, show significant effects on phosphoryl transfer and downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271 mutations increase the average number of supercoils that are removed, enhancing the processivity of supercoil unwinding. These measurements support the proposal that the processivity of supercoil unwinding can be regulated by electrostatic interactions between the enzyme and the mobile DNA phosphate backbone.