DNA Topoisomerase III from the Hyperthermophilic Archaeon Sulfolobus solfataricus with Specific DNA Cleavage Activity

Abstract

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. Topoisomerases are ubiquitous enzymes that participate in nearly all cellular activities involving DNA transactions (5, 31). Based on their catalytic mechanisms, topoisomerases are classified into two classes: type I enzymes, which generate a transient break on one DNA strand to allow the passage of the intact strand with a linking change of one in a single reaction cycle, and type II enzymes, which introduce a transient doublestranded break to allow the passage of another duplex DNA segment with a change in linking number by steps of two (5, 30). Each class of topoisomerases is further divided into two structurally and mechanistically distinct subfamilies on the basis of the polarity of enzyme attachment to the broken strands (i.e., IA, IB, IIA, and IIB). A cellular organism possesses multiple forms of topoisomerases. For example, Escherichia coli has two type IA enzymes (topoisomerases I and III) and two type IIA enzymes (gyrase and topoisomerase IV) (30). Humans carry two enzymes of each of the subfamilies IA (topoisomerases III␣ and III␤), IB (topoisomerase I and mitochondrial topoisomerase I), and IIA (topoisomerases II␣ and II␤) (31). Sulfolobus, a group of hyperthermophilic archaea, possesses two type IA enzymes (putative topoisomerase I, identified by gene annotation, and reverse gyrase) and a type IIB enzyme (topoisomerase VI) (2,8,10). It appears that all organisms have at least one type IA topoisomerase (31).E. coli DNA topoisomerase I is the most extensively studied representative of type IA enzymes. The E. coli enzyme is composed structurally of an N-terminal transesterification domain and a C-terminal DNA binding domain (1,17). The N-terminal domain has the active site tyrosine for DNA cleavage and is capable of forming a covalent adduct with single-stranded DNA. But this domain cannot relax negatively supercoiled DNA. The C-terminal domain consists of five copies of a zinc ribbon motif and is suspected to interact with the passing strand of DNA in the relaxation of negatively supercoiled DNA (1, 12). Each of the first three zinc ribbon motifs from the N terminus in this domain contains four cysteine residues at required sites and is likely to bind zinc (28). The fourth and fifth motifs have one and no cysteine residue, respectively. Sequence analysis of type IA topoisomerase-encoding genes has revealed a significant variation with respect to the copy number of zinc ribbon motifs (0 to 5) as well as the number of tetracysteine motifs in the enzymes (12). These data suggest that zinc ribbon motifs may not be required for DNA relaxation by at least some type IA topoisomerases. This suggestion is consistent with reports that zinc binding is not necessary for the relaxation activity of topoisomerases from Mycobacterium smegmatis and Thermotoga maritima (3,29).Although type IA topoisomerases share considerable seq...

Oligonucleotide cleavage and rejoining by topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus: temperature dependence and strand annealing‐promoted DNA religation

“…Sso topo III is sequence-specific in oligonucleotide cleavage, showing the strictest requirement for bases at positions −6 to −3 (Dai et al, 2003). In this study, we found that complementarity to a region from −6 to +2 in the C strand was important for annealing-promoted religation of the cleaved strand by the enzyme.…”

“…It was shown in the previous study that oligonucleotide cleavage by Sso topo III was not detectable at 0 ° C, very low but measurable at 37 ° C, and peaked at 65-75 ° C (Dai et al ., 2003). However, this pattern of temperature dependence was subsequently found to result from nucleic acid contamination in the enzyme preparation.…”

“…The location of this region in the NC strand did not appear to be crucial, suggesting that complementarity of the NC strand to no sequences outside the region between −6 and +2 in the C strand was absolutely required for annealing-promoted religation. It is worth noting that the region in the C strand important for the annealing-promoted strand religation is not identical to, though overlaps with, that most conserved for strand cleavage by Sso topo III (−6 to −3) (Dai et al, 2003).…”

“…The exact role of reverse gyrase is unclear, but it may function to maintain DNA in a relaxed to positively supercoiled state (Forterre, 2002). The other type IA enzyme has recently been proposed to be topoisomerase III based on sequence and cleavage site analyses (Dai et al ., 2003). Given the DNA topology in hyperthermophilic archaea, topoisomerase III is less likely than reverse gyrases to be involved in regulating DNA supercoiling.…”

“…Because of their thermostability, thermophilic type IA topoisomerases offer a unique model for studying the mechanism of this class of topoisomerases as well as its adaptation to high temperature. We have recently characterized a topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus ( Sso topo III) (Dai et al ., 2003). The enzyme is capable of efficiently relaxing negatively supercoiled DNA at 75 ° C. In the present study, we show that Sso topo III exhibits distinctly different patterns of temperature dependence in oligonucleotide cleavage and religation, and facilitates DNA annealing.…”