Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.In all living cells the induction of DNA damage activates an extremely complex network of events involving every cell process from DNA replication and transcription to cell division, protein synthesis and degradation, and, eventually, cell death (1). A major challenge in cell biology is elucidating how these events are regulated and connected. Archaea are helpful model systems for studying pathways of DNA transactions (replication, transcription, recombination, and repair) that are thought of as simplified and ancient versions of the eukaryal ones (reviewed in Refs. 2 and 3). However, to date the response to DNA damage has been poorly investigated in Archaea.Genome sequencing has revealed the presence of archaeal genes homologous to components of the eukaryal nucleotide excision repair pathway, which is involved in the repair of UV-induced DNA lesions (4, 5). We have shown previously that the exposure of Sulfolobus solfataricus to UV light and the intercalating agent actinomycin D elicits a DNA damage response with features essentially conserved in all three domains of life, including growth arrest and transcriptional induction of nucleotide excision repair genes. UV light and actinomycin D also regulate the genes encoding two DNA-binding proteins affecting DNA conformation, namely Sul7d and Smj12 (6). Sul7d is an abundant chromosomal protein that induces DNA bending, compaction, and negative supercoiling (7). In contrast, Smj12 induces positive supercoiling (8). Although the reasons for this regulation are currently only matters of spec...

Reverse gyrase is a peculiar DNA topoisomerase, specific of thermophilic microorganisms, which induces positive supercoiling into DNA molecules in an ATP-dependent reaction. It is a modular enzyme and comprises an N-terminal helicase-like module fused to a C-terminal topoisomerase IA-like domain. The exact molecular mechanism of this unique reaction is not understood, and a fundamental mechanistic question is how its distinct steps are coordinated. We studied the cross-talk between the components of this molecular motor and probed communication between the DNA-binding sites and the different activities (DNA relaxation, ATP hydrolysis and positive supercoiling). We show that the isolated ATPase and topoisomerase domains of reverse gyrase form specific physical interactions, retain their own DNA binding and enzymatic activities, and when combined cooperate to achieve the unique ATP-dependent positive supercoiling activity. Our results indicate a mutual effect of both domains on all individual steps of the reaction. The C-terminal domain shows ATP-independent topoisomerase activity, which is repressed by the N-terminal domain in the full-length enzyme; experiments with the isolated domains showed that the C-terminal domain has stimulatory influence on the ATPase activity of the N-terminal domain. In addition, the two domains showed a striking reciprocal thermostabilization effect.