Claire Bouthier de la Tour

The Deinococcus radiodurans SMC protein is dispensable for cell viability yet plays a role in DNA folding

Deinococcus radiodurans contains a highly condensed nucleoid that remains to be unaltered following the exposure to high doses of γ-irradiation. Proteins belonging to the structural maintenance of chromosome protein (SMC) family are present in all organisms and were shown to be involved in chromosome condensation, pairing, and/or segregation. Here, we have inactivated the smc gene in the radioresistant bacterium D. radiodurans, and, unexpectedly, found that smc null mutants showed no discernible phenotype except an increased sensitivity to gyrase inhibitors suggesting a role of SMC in DNA folding. A defect in the SMC-like SbcC protein exacerbated the sensitivity to gyrase inhibitors of cells devoid of SMC. We also showed that the D. radiodurans SMC protein forms discrete foci at the periphery of the nucleoid suggesting that SMC could locally condense DNA. The phenotype of smc null mutant leads us to speculate that other, not yet identified, proteins drive the compact organization of the D. radiodurans nucleoid.

Cloning and sequencing of the gene coding for topoisomerase I from the extremely thermophilic eubacterium, Thermotoga maritima

A 2767 bp fragment containing a gene coding for a topoisomerase I from the extremely thermophilic eubacterium Thermotoga maritima (Tm TopA) has been cloned and sequenced. The protein is composed of 633 amino acids with a calculated molecular mass of 72,695 Da. It shares significant similarity with the topoisomerases I of mesophilic eubacteria. The highest score is obtained with Bacillus subtilis (44% identity); in particular, T. maritima and B. subtilis possess an insertion of 7-8 amino acids in the vicinity of the active site, that is absent in topoisomerases of other organisms. A specific feature of T. maritima topoisomerase I is its low cysteine content compared to its mesophilic homologs. It contains 5 cysteine residues, of which 4 could constitute a zinc finger motif. Finally, analysis of the regions flanking the gene reveals that Tm TopA is surrounded by two other ORFs, suggesting the occurrence of a polycistronic transcriptional unit.

ATP-independent DNA topoisomerase from Fervidobacterium islandicum

Thermotogales are thermophilic eubacteria belonging to a very slowly evolving branch in the eubacterial tree. In this report, we describe the purification and characterization of an ATP-independent DNA topoisomerase from the Thermotogale, Fervidobacterium islandicum. The enzyme, a monomer of about 75 kDa, is a type I DNA topoisomerase sharing many properties with the other bacterial topoisomerases I: it absolutely requires Mg2+ for activity, relaxes negatively but not positively supercoiled DNA and is inhibited by single-stranded M13 DNA and spermidine. A feature of the F. islandicum ATP-independent DNA topoisomerase I is its thermophily. The optimal temperature for the enzymatic activity is 75 degrees C. Studies about thermostability show that the enzyme is more stable when incubated undiluted in the storage buffer. In these conditions, 60% activity was retained after a 30 min preincubation at 75 degrees C.

Mutational Analysis of the Helicase-like Domain of Thermotoga maritima Reverse Gyrase

Reverse gyrase is a unique type IA topoisomerase that is able to introduce positive supercoils into DNA in an ATP-dependent process. ATP is bound to the helicase-like domain of the enzyme that contains most of the conserved motifs found in helicases of the SF1 and SF2 superfamilies. In this paper, we have investigated the role of the conserved helicase motifs I, II, V, VI, and Q by generating mutants of the Thermotoga maritima reverse gyrase. We show that mutations in motifs I, II, V, and VI completely eliminate the supercoiling activity of reverse gyrase and that a mutation in the Q motif significantly reduces this activity. Further analysis revealed that for most mutants, the DNA binding and cleavage properties are not significantly changed compared with the wild type enzyme, whereas their ATPase activity is impaired. These results clearly show that the helicase motifs are tightly involved in the coupling of ATP hydrolysis to the topoisomerase activity. The zinc finger motif located at the N-terminal end of reverse gyrases was also mutated. Our results indicate that this motif plays an important role in DNA binding.

[12] Reverse gyrases from bacteria and archaea

Publisher Summary Among the enzymes that are responsible for shaping DNA, reverse gyrases are particularly interesting because of their structure and the unique reaction they catalyze: the formation of positive supercoils in circular DNA. Reverse gyrases are classified as type I topoisomerases because they change the DNA linking number by increments of one. Remarkably, this peculiar enzyme appears widely distributed in both the archaeal and bacterial kingdoms, but seems restricted to hyperthermophilic strains. Its presence in eukaryotes has been postulated, although without strong evidence so far. Furthermore, reverse gyrase activity is detectable in extracts of all hyperthermophilic species so far tested, provided that it is not masked by another prominent topoisomerase. This distribution makes the enzyme a good marker of thermophily. The biological function of reverse gyrases is still a matter of conjecture. However, since reverse gyrases seem essential in extremely thermophilic organisms, it was postulated that these enzymes participate in the stabilization of the DNA double helix. This chapter describes the purification of reverse gyrases from their natural hosts and from recombinant Escherichia coli strains, in addition to the various assays developed to test their biochemical properties. Finally, the main structural features of these enzymes and their putative mechanism are discussed, together with their possible in vivo functions.

DNA Topoisomerases I From Thermophilic Bacteria: Cloning and Sequencing of the DNA Topoisomerase I from Fervidobacterium islandicum

Summary The gene encoding the DNA topoisomerase I from the thermophilic bacterium Fervidobacterium islandicum has been cloned by using oligonucleotides derived from internal microsequencing of the purified protein ( Bouthier de la Tour et al., 1993). The corresponding nucleotide sequence has been determined, revealing an open reading frame of 2097 nucleotides. The deduced amino acid sequence (699 residues) clearly showed that the gene belongs to E. coli TopA family, and has 53% identity with Thermotoga maritima TopA ( Bouthier de la Tour et al., 1995). Comparison of the F. islandicum sequence to the topoisomerase I sequences of other species revealed several interesting features: (1) a putative N-terminal tail of 33 amino acids, whose function is unknown. (2) a 8 amino acids insertion close to the active site tyrosine, shared by 3 other bacterial TopA sequences. (3) a unique putative zinc finger that is conserved in 7 out of 9 sequences. However, no significant feature linked to thermophily could distinguish the two thermotogales topoisomerases from their mesophilic counterparts. Finally, the analysis of the gene organization in the vicinity of the TopA locus in different bacterial species showed that it was not conserved.

Deinococcus radiodurans' SRA-HNH domain containing protein Shp (Dr1533) is involved in faithful genome inheritance maintenance following DNA damage

BACKGROUND Deinococcus radiodurans R1 (DR) survives conditions of extreme desiccation, irradiation and exposure to genotoxic chemicals, due to efficient DNA breaks repair, also through Mn2+ protection of DNA repair enzymes. METHODS Possible annotated domains of the DR1533 locus protein (Shp) were searched by bioinformatic analysis. The gene was cloned and expressed as fusion protein. Band-shift assays of Shp or the SRA and HNH domains were performed on oligonucleotides, genomic DNA from E. coli and DR. shp knock-out mutant was generated by homologous recombination with a kanamycin resistance cassette. RESULTS DR1533 contains an N-terminal SRA domain and a C-terminal HNH motif (SRA-HNH Protein, Shp). Through its SRA domain, Shp binds double-strand oligonucleotides containing 5mC and 5hmC, but also unmethylated and mismatched cytosines in presence of Mn2+. Shp also binds to Escherichia coli dcm+ genomic DNA, and to cytosine unmethylated DR and E. coli dcm- genomic DNAs, but only in presence of Mn2+. Under these binding conditions, Shp displays DNAse activity through its HNH domain. Shp KO enhanced >100 fold the number of spontaneous mutants, whilst the treatment with DNA double strand break inducing agents enhanced up to 3-log the number of survivors. CONCLUSIONS The SRA-HNH containing protein Shp binds to and cuts 5mC DNA, and unmethylated DNA in a Mn2+ dependent manner, and might be involved in faithful genome inheritance maintenance following DNA damage. GENERAL SIGNIFICANCE Our results provide evidence for a potential role of DR Shp protein for genome integrity maintenance, following DNA double strand breaks induced by genotoxic agents.