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

Abstract

Type IB topoisomerases unwind positive and negative DNA supercoils and play a key role in removing supercoils that would otherwise accumulate at replication and transcription forks. An interesting question is whether topoisomerase activity is regulated by the topological state of the DNA, thereby providing a mechanism for targeting the enzyme to highly supercoiled DNA domains in genomes. The type IB enzyme from variola virus (vTopo) has proven to be useful in addressing mechanistic questions about topoisomerase function because it forms a reversible 3′-phosphotyrosyl adduct with the DNA backbone at a specific target sequence (5′-CCCTT-3′) from which DNA unwinding can proceed. We have synthesized supercoiled DNA minicircles (MCs) containing a single vTopo target site that provides highly defined substrates for exploring the effects of supercoil density on DNA binding, strand cleavage and ligation, and unwinding. We observed no topological dependence for binding of vTopo to these supercoiled MC DNAs, indicating that affinity-based targeting to supercoiled DNA regions by vTopo is unlikely. Similarly, the cleavage and religation rates of the MCs were not topologically dependent, but topoisomers with low superhelical densities were found to unwind more slowly than highly supercoiled topoisomers, suggesting that reduced torque at low superhelical densities leads to an increased number of cycles of cleavage and ligation before a successful unwinding event. The K271E charge reversal mutant has an impaired interaction with the rotating DNA segment that leads to an increase in the number of supercoils that were unwound per cleavage event. This result provides evidence that interactions of the enzyme with the rotating DNA segment can restrict the number of supercoils that are unwound. We infer that both superhelical density and transient contacts between vTopo and the rotating DNA determine the efficiency of supercoil unwinding. Such determinants are likely to be important in regulating the steady-state superhelical density of DNA domains in the cell.

AP-Endonuclease 1 Accelerates Turnover of Human 8-Oxoguanine DNA Glycosylase by Preventing Retrograde Binding to the Abasic-Site Product

“…Such discrepancies between experiments that employ excess enzyme over DNA and the reverse condition of excess DNA have been observed previously with hOGG1 14 and other enzymes. 36 The most likely explanation is that with excess DNA binding sites the target search time has become ratelimiting, which is supported by the observation that the presteady-state burst rate gradually increases as the hOGG1 concentration increases (Figure S1B and Table S1). In addition, our previous studies have indicated that the target search time of hOGG1 can be adversely affected by the presence of nonspecific DNA sequences.…”

RNA topoisomerase is prevalent in all domains of life and associates with polyribosomes in animals

“…In addition, several Type IB topoisomerases can cleave ribonucleotides incorporated in DNA ( 45 , 46 ), hinting that they may have RNA topoisomerase activity. Our screening showed that three Type IB DNA topoisomerases, human topoisomerase I (Top1) ( 47 ), human mitochondrial topoisomerase (Top1mt) ( 48 ), and Variola virus topoisomerase ( 49 ) failed to convert the RNA circle to knot (Supplementary Figure S2A-C). However, we cannot exclude the possibility that these enzymes may relax supercoiled dsRNA similar to their actions on dsDNA.…”

SAMHD1 is a single-stranded nucleic acid binding protein with no active site-associated nuclease activity

“…To prepare a duplex substrate containing a single-stranded DNA overhang, a region of the pMC454 plasmid ( 23 ) containing an Nt.BbvCI site and SalI site was amplified by PCR using oligonucleotide primers. Sequential digestion of the PCR product by SalI and Nt.BbvCI produced a 272 bp duplex with a 23 nt ssDNA 5′ overhang on one end.…”