Human telomeres are protected from DNA damage by a nucleoprotein complex that includes the repeat-binding factor TRF2. Here, we report that TRF2 regulates the 5' exonuclease activity of its binding partner, Apollo, a member of the metallo-beta-lactamase family that is required for telomere integrity during S phase. TRF2 and Apollo also suppress damage to engineered interstitial telomere repeat tracts that were inserted far away from chromosome ends. Genetic data indicate that DNA topoisomerase 2alpha acts in the same pathway of telomere protection as TRF2 and Apollo. Moreover, TRF2, which binds preferentially to positively supercoiled DNA substrates, together with Apollo, negatively regulates the amount of TOP1, TOP2alpha, and TOP2beta at telomeres. Our data are consistent with a model in which TRF2 and Apollo relieve topological stress during telomere replication. Our work also suggests that cellular senescence may be caused by topological problems that occur during the replication of the inner portion of telomeres.

DNA damage induced by reactive carbonyls (mainly methylglyoxal and glyoxal), called DNA glycation, is quantitatively as important as oxidative damage. DNA glycation is associated with increased mutation frequency, DNA strand breaks, and cytotoxicity. However, in contrast to guanine oxidation repair, how glycated DNA is repaired remains undetermined. Here, we found that the parkinsonism-associated protein DJ-1 and its bacterial homologs Hsp31, YhbO, and YajL could repair methylglyoxal- and glyoxal-glycated nucleotides and nucleic acids. DJ-1-depleted cells displayed increased levels of glycated DNA, DNA strand breaks, and phosphorylated p53. Deglycase-deficient bacterial mutants displayed increased levels of glycated DNA and RNA and exhibited strong mutator phenotypes. Thus, DJ-1 and its prokaryotic homologs constitute a major nucleotide repair system that we name guanine glycation repair.

Reverse gyrase is a type I DNA topoisomerase able to positively supercoil DNA and is found in thermophiic archaebacteria and eubacteria. The gene coding for this protein was cloned from Sulfolobus acidocaldarius DSM 639. Analysis of the 1247-amino acid sequence and comparison of it with available sequence data suggest that reverse gyrase is constituted of two distinct domains: (i) a C-terminal domain of -630 amino acids clearly related to eubacterial topoisomerase I (Escherichia coli topA and topB gene products) and to Saccharomyces cerevisiae top3; (ii) an N-terminal domain without any similarity to other known topoisomerases but containing several helicase motifs, including an ATP-binding site. These results are consistent with those from our previous mechanistic studies of reverse gyrase and suggest a model in which positive supercoiling is driven by the concerted action of helicase and topoisomerase in the same polypeptide: this constitutes an example of a composite gene formed by a helicase domain and a topoisomerase domain.It is today well established that topoisomerases play a crucial role in DNA structure and function (1, 2). These enzymes seem to act in two ways: (i) they are able to solve the topological problems intrinsic to the DNA double helix during replication, transcription, recombination, or chromatin condensation and decondensation; (ii) some topoisomerases have exploited the circular or pseudocircular (chromatin loops) structure of the genetic material to introduce stress into DNA by means of supercoiling. The enzymes specialized in the production of superhelical turns in a circular DNA were named "gyrases" (3, 4). Among these, the best characterized enzyme is the eubacterial gyrase, a type II topoisomerase that uses the energy of ATP hydrolysis to maintain the in vivo level of negative supercoiling of the bacterial chromosome (5). Several years ago, another kind of DNA supercoiling activity was discovered in hyperthermophilic archaebacteria (6) and was called "reverse gyrase." We have shown that reverse gyrase is widely distributed in various archaebacterial families and also in thermophilic eubacteria (7,8). This enzyme has the specific ability to catalyze in vitro positive supercoiling of a closed circular DNA at high temperature (9). Surprisingly, reverse gyrase turned out to be a type I topoisomerase (9) and is the only topoisomerase I that depends on ATP and can catalyze DNA supercoiling. The biological function of reverse gyrase is still unclear, but we found that positive supercoiling occurs in vivo (10). The enzyme from Sulfolobus acidocaldarius is composed of a single polypeptide of apparent molecular mass of -130,000 (11). Mechanistic studies (12) indicated that reverse gyrase transiently cleaves a single DNA strand, forming a covalent link with the 5' end of the broken strand, as described for eubacterial topoisomerase I (13). In addition, we have shown that stoichiometric binding of the enzyme changes the DNA conformation, presumably by unwinding the double helix (12). We ...

DNA Topoisomerases are essential to resolve topological problems during DNA metabolism in all species. However, the prevalence and function of RNA topoisomerases remain uncertain. Here, we show that RNA topoisomerase activity is prevalent in Type IA topoisomerases from bacteria, archaea, and eukarya. Moreover, this activity always requires the conserved Type IA core domains and the same catalytic residue used in DNA topoisomerase reaction; however, it does not absolutely require the non-conserved carboxyl-terminal domain (CTD), which is necessary for relaxation reactions of supercoiled DNA. The RNA topoisomerase activity of human Top3β differs from that of Escherichia coli topoisomerase I in that the former but not the latter requires the CTD, indicating that topoisomerases have developed distinct mechanisms during evolution to catalyze RNA topoisomerase reactions. Notably, Top3β proteins from several animals associate with polyribosomes, which are units of mRNA translation, whereas the Top3 homologs from E. coli and yeast lack the association. The Top3β-polyribosome association requires TDRD3, which directly interacts with Top3β and is present in animals but not bacteria or yeast. We propose that RNA topoisomerases arose in the early RNA world, and that they are retained through all domains of DNA-based life, where they mediate mRNA translation as part of polyribosomes in animals.

Repairing DNA double-strand breaks (DSBs) by non-homologous end-joining (NHEJ) requires multiple proteins to recognize and bind DNA ends, process them for compatibility, and ligate them together. We constructed novel DNA substrates for single-molecule nano-manipulation allowing us to mechanically detect, probe, and rupture in real-time DSB synapsis by specific human NHEJ components. DNA-PKcs and Ku allow DNA end synapsis on the 100 ms timescale, and addition of PAXX extends this lifetime to ~2s. Further addition of XRCC4, XLF and Ligase IV resulted in minute-scale synapsis and led to robust repair of both strands of the nanomanipulated DNA. The energetic contribution of the different components to synaptic stability is typically on the scale of a few kCal/mol. Our results define assembly rules for NHEJ machinery and unveil the importance of weak interactions, rapidly ruptured even at sub-picoNewton forces, in regulating this multicomponent chemomechanical system for genome integrity.

A topoisomerase able to introduce positive supercoils in a closed circular DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. This enzyme, fully active at 75 degrees C, performed in vitro positive supercoiling either from negatively supercoiled, or from relaxed DNA in a catalytic reaction. In the presence of polyethylene glycol (PEG 6000), this reaction became very fast and highly processive, and the product was positively supercoiled DNA with a high superhelical density (form I+). Very low (5 ‐ 10 micromoles) ATP concentrations were sufficient to support full supercoiling; the nonhydrolyzable analogue adenosine‐5′ ‐0‐(3‐thiotriphosphate) also sustained the production of positive supercoils, but to a lesser extent, suggesting that ATP hydrolysis was necessary for efficient activity. Nevertheless, low residual of positive supercoiling occurred, even in the absence of ATP, when the substrate was negatively supercoiled. Finally, the different ATP‐driven topoisomerizations observed, i.e., relaxation of negative supercoils and positive supercoiling, in all cases increased the linking number of DNA in steps of 1, suggesting the action of a type I, rather than a type II topoisomerase.=

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...

Investigation of the presence of a reverse gyrase-like activity in archaebacteria revealed wide distribution of this activity in hyperthermophilic species, including methanogens and sulfur-dependent organisms. In contrast, no reverse gyrase activity was detected in mesophilic and moderately thermophilic organisms, which exhibited only an ATP-independent activity of DNA relaxation. These results suggest that the presence of reverse gyrase in archaebacteria is tightly linked to the high growth temperatures of these organisms. With respect to antigenic properties, the enzyme appeared similar among members of the genus Sulfolobus. In contrast, no close antigenic relatedness was found between the reverse gyrase of members of the order Sulfolobales and that of the other hyperthermophilic organisms.On the basis of partial rRNA sequence comparisons, archaebacteria have been divided into two major branches: the sulfur-dependent and the methanogenic plus halophilic archaebacteria, including the genus Thermoplasma (37, 38). Thermophilic archaebacteria are found in these two branches; indeed, although most of them are clustered in the first group (32), thermophilic and hyperthermophilic species are also found in the group of methanogens. Likewise, Archaeoglobus, a genus that occupies an intermediate position between the two major groups, is also an extremely thermophilic archaebacterium, with an optimum growth temperature at 830C (1,29,31,39).Biochemical mechanisms allowing growth at very high temperatures are still not well known. A few years ago, a singular topoisomerase, the reverse gyrase, capable of introducing positive supercoils into closed circular plasmid DNA was isolated from two sulfur-dependent hyperthermophilic archaebacteria, Sulfolobus acidocaldarius (9, 17) and Desulfurococcus amylolyticus (28). The enzyme purified from these two organisms is made of a single polypeptide of about 120 to 135 kDa (21,23,28). It is the only known topoisomerase that exhibits an activity of reverse gyration (positive supercoiling of DNA) per se and that is both a type I and an ATP-dependent topoisomerase (9, 23). Furthermore, the DNA of the viruslike particle SSV1 (present in Sulfolobus sp. strain B12) was found to be positively supercoiled after extraction (22), suggesting that the reverse gyrase activity takes place in vivo. From these results, it seemed interesting to determine whether positive supercoiling of DNA was essential for life at high temperatures and whether the presence of reverse gyrase reflected a property of a phylogenetically restricted group of organisms. In the latter case, analysis of the distribution of reverse gyrase within the archaebacterial kingdom could constitute a tool to confirm the phylogenetic position of some archaebacteria.We have examined the presence of reverse gyrase in a large variety of strains, including sulfidogens and methanogens. We found the reverse gyrase activity (i.e., ATP- (about 2 x 10-3 U), the mixture was incubated for 15 min at 75°C. The reaction was stopped by quick cooling an...