Diverse Energetic Effects of Charge Reversal Mutations of Poxvirus Topoisomerase IB

Abstract

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 site of cleavage must rotate around the helical axis, requiring that interactions with the enzyme must break and reform multiple times during the course of removing supercoils. A crystal structure of variola virus type IB topoisomerase (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, with little effect on enzyme-DNA binding, or the processivity of supercoil unwinding. 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 two mutations of K271, which interacts with mobile region even further from the site of covalent linkage, show significant effects not only on phosphoryl transfer, but also downstream DNA strand positioning. Moreover, supercoil unwinding measurements indicate that the K271A and K271E mutations increase the average number of supercoils that are removed during the lifetime of the covalent complex, 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.

Variola Type IB DNA Topoisomerase: DNA Binding and Supercoil Unwinding Using Engineered DNA Minicircles

“…Although all MCs migrated as single bands during agarose gel electrophoresis, they consisted of a Gaussian distribution of topoisomers when electrophoresis was performed using 5% polyacrylamide gels in the presence of 10 mM MgCl 2 , or using a 6% native gel in the presence of chloroquine (Figure 2 and Figure S1 of the Supporting Information ). 20 , 22 , 24 The 454 bp minicircle that lacked the specific site (MC ns ), as well as its corresponding highly supercoiled form MC ns* , followed the same topological distributions as MC sp and MC sp* .…”

“…Previous studies indicated that cationic residues on vTopo interact with the negatively charged DNA backbone of the rotating DNA segment and impact the number of supercoils that are released per cleavage event. 21 , 22 , 36 A previously characterized vTopo mutant (K271E) was known to increase the number of supercoils that were removed per cleavage event and was a desirable mutant to test using MC sp2 . 22 Indeed, relaxation experiments with MC sp2 showed slower cleavage and fewer intermediates with K271E than with wild-type vTopo (compare panels B and C of Figure 7 ).…”

“… 21 , 22 , 36 A previously characterized vTopo mutant (K271E) was known to increase the number of supercoils that were removed per cleavage event and was a desirable mutant to test using MC sp2 . 22 Indeed, relaxation experiments with MC sp2 showed slower cleavage and fewer intermediates with K271E than with wild-type vTopo (compare panels B and C of Figure 7 ). The numerical simulations indicated that the probability of completely unwinding the substrate pool by K271E was increased compared to that with wild-type vTopo (for K271E, k s→p / k s→I1 = 7.7 ± 1.5 and k s→p / k s→I2 = 15.1 ± 5.1), but unwinding of the two intermediate pools was similar (for K271E, k I1→p / k lig = 0.017 ± 0.0094 and k I2→p / k lig = 0.013 ± 0.017).…”

“…vTopo is known to make interactions with the rotating DNA segment downstream of the cleavage site, and such interactions have been previously implicated in supercoil unwinding. 16 , 22 Crystal structures show that residue K271 sits within helix 10 of vTopo and could contribute to the positioning of the DNA segment downstream of the cleavage site to facilitate religation. 36 We anticipated that reversing the charge on this amino acid might decrease the probability for religation, resulting in an increased k s→p / k s→i ratio.…”

“… 20 , 21 Moreover, the number of supercoils that are removed during the lifetime of the covalent intermediate is influenced by interactions between the enzyme and the DNA and the intrinsic superhelical torque. 21 , 22 …”