Oligonucleotide cleavage and rejoining by topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus: temperature dependence and strand annealing-promoted DNA religation.
Topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus (Sso topo III) is optimally active in DNA relaxation at 75 degrees C. We report here that Sso topo III-catalysed DNA cleavage and religation differed significantly in temperature dependence: the enzyme was most active in cleaving ssDNA containing a cleavage site at 25-50 degrees C, but was efficient in rejoining the cleaved DNA strand only at higher temperatures (e.g. > or = 45 degrees C). The failure of Sso topo III to rejoin the cleaved DNA strand efficiently appeared to be responsible for the inability of the enzyme to relax negatively supercoiled DNA at low temperature (e.g. 25 degrees C). Intriguingly, Sso topo III facilitated DNA annealing although it showed higher affinity for ssDNA than for dsDNA. Religation of the DNA strand cleaved by Sso topo III was drastically enhanced when the DNA was allowed to anneal to a complementary non-cleaved oligonucleotide, presumably as a result of destabilization of the interaction between the enzyme and the cleaved strand through the formation of duplex DNA. A region in the non-cleaved strand corresponding to a sequence containing six bases on the 5' side and two bases on the 3' side of the cleavage site in the cleaved strand was crucial to the annealing-promoted religation. However, the annealing-promoted religation was relatively insensitive to mismatches in this region and the region conserved for oligonucleotide cleavage, except for that at the 5' end of the broken strand. These results suggest that Sso topo III is well suited for a role in DNA rewinding, whether it leads to homoduplex or heteroduplex formation.
Chen
Liang
L
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, China.
Huang
Li
L
D019843:Archaeal Proteins / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D004264:DNA Topoisomerases, Type I / Q000235:genetics / Q000378:metabolism*; D019641:DNA, Archaeal / Q000378:metabolism*; D004278:DNA, Superhelical / Q000378:metabolism; D004795:Enzyme Stability; D019848:Gene Expression Regulation, Archaeal; D006358:Hot Temperature; D009841:Oligonucleotides / Q000378:metabolism*; D048229:Sulfolobus solfataricus / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism
D019843:Archaeal Proteins; D019641:DNA, Archaeal; D004278:DNA, Superhelical; D009841:Oligonucleotides; D004264:DNA Topoisomerases, Type I