Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene dele...Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was ,viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different tran- scriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.展开更多
In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is ...In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.展开更多
基金supported by the National Natural Science Foundation of China(Nos.30921065,30730003 and 30870058) to L.Huangthe Danish Research Council for Technology and Production(No.274-07-0116)
文摘Topoisomerase III （topo III）, a type IA topoisomerase, is widespread in hyperthermophilic archaea. In order to interrogate the in vivo role of archaeal topo III, we constructed and characterized a topo III gene deletion mutant of Sulfolobus islandicus. The mutant was ,viable but grew more slowly than the wild-type strain, especially in a nutrient-poor medium. Flow cytometry analysis revealed changes of the mutant in growth cycle characteristics including an increase in proportion of cells containing either more than two genome equivalents or less than one genome equivalent in exponentially-growing cultures. As shown by fluorescence microscopy, a fraction of mutant cells in the cultures were drastically enlarged, and at least some of the enlarged cells were apparently capable of resuming cell division. The mutant also shows a different tran- scriptional profile from that of the wild-type strain. Our results suggest that the enzyme may serve roles in chromosomal segregation and control of the level of supercoiling in the cell.
基金supported by the grants from the National Natural Science Foundation of China (Nos. 3093002, 31170072 and 31470184 to Y.S.)
文摘In archaea, the HEL308 homolog Hel308a（or Hjm） is implicated in stalled replication fork repair. The biochemical properties and structures of Hjm homologs are well documented, but in vivo mechanistic information is limited. Herein, a structure-based functional analysis of Hjm was performed in the genetically tractable hyperthermophilic archaeon, Sulfolobus islandicus. Results showed that domain V and residues within it, which affect Hjm activity and regulation, are essential and that the domain V-truncated mutants and sitedirected mutants within domain V cannot complement hjm chromosomal deletion. Chromosomal hjm deletion can be complemented by ectopic expression of hjm under the control of its native promoter but not an artificial arabinose promoter. Cellular Hjm levels are kept constant under ultraviolet（UV） and methyl methanesulfonate（MMS） treatment conditions in a strain carrying a plasmid to induce Hjm overexpression. These results suggest that Hjm expression and activity are tightly controlled, probably at the translational level.
