Reverse Gyrase Recruitment to DNA after UV Light Irradiation in Sulfolobus solfataricus

105

The DNA binding affinity of Alba, a chromatin protein of the archaeon Sulfolobus solfataricus P2, is regulated by acetylation of lysine 16. Here we identify an acetyltransferase that specifically acetylates Alba on this residue. The effect of acetylation is to lower the affinity of Alba for DNA. Remarkably, the acetyltransferase is conserved not only in archaea but also in bacteria where it appears to play a role in metabolic regulation. Therefore, our data suggest that S. solfataricus has co-opted this bacterial regulatory system to generate a rudimentary form of chromatin regulation.

Section: Discussionmentioning

confidence: 99%

Sir2 and the Acetyltransferase, Pat, Regulate the Archaeal Chromatin Protein, Alba

Marsh

Peak‐Chew

Bell

2005

105

“…Eight liters of cell culture were grown at 80°C as described previously (22) until exponential phase (0.4 -0.6 A 600 ). Cells were harvested by centrifugation, and soluble protein extract was prepared as described (17). Soluble extract was incubated for 30 min at 37°C with 20 units/ml Benzonase (Novagen) dialyzed against heparin buffer (20 mM Tris-HCl, pH 7.0) and loaded onto a heparin column (HiPrep 16/10 heparin FF, GE Healthcare).…”

Section: Methodsmentioning

confidence: 99%

“…Several results suggest that reverse gyrase might participate in the cell response to DNA damage. In the crenarchaeon Sulfolobus solfataricus, reverse gyrase is recruited to DNA after UV irradiation (17), interacts with the single strand-binding protein (SSB), and the translesion DNA polymerase, PolY (18,19), and is degraded after treatment with methyl methane sulfonate (MMS) (20). Reverse gyrase structure and its involvement in genome stability are reminiscent of the evolutionary conserved complexes comprising topoisomerase III and members of the RecQ helicase family, which have essential functions in recombination and repair (for a recent review, see Ref.…”

mentioning

confidence: 99%

The Archaeal Topoisomerase Reverse Gyrase Is a Helix-destabilizing Protein That Unwinds Four-way DNA Junctions

Valenti

Perugino

Varriale

et al. 2010

Self Cite

Four-way junctions are non-B DNA structures that originate as intermediates of recombination and repair (Holliday junctions) or from the intrastrand annealing of palindromic sequences (cruciforms). These structures have important functional roles but may also severely interfere with DNA replication and other genetic processes; therefore, they are targeted by regulatory and architectural proteins, and dedicated pathways exist for their removal. Although it is well known that resolution of Holliday junctions occurs either by recombinases or by specialized helicases, less is known on the mechanisms dealing with secondary structures in nucleic acids. Reverse gyrase is a DNA topoisomerase, specific to microorganisms living at high temperatures, which comprises a type IA topoisomerase fused to an SF2 helicase-like module and catalyzes ATP hydrolysis-dependent DNA positive supercoiling. Reverse gyrase is likely involved in regulation of DNA structure and stability and might also participate in the cell response to DNA damage. By applying FRET technology to multiplex fluorophore gel imaging, we show here that reverse gyrase induces unwinding of synthetic four-way junctions as well as forked DNA substrates, following a mechanism independent of both the ATPase and the strandcutting activity of the enzyme. The reaction requires high temperature and saturating protein concentrations. Our results suggest that reverse gyrase works like an ATP-independent helix-destabilizing protein specific for branched DNA structures. The results are discussed in light of reverse gyrase function and their general relevance for protein-mediated unwinding of complex DNA structures.Four-way junctions are complex DNA structures that play a major biological role as intermediates in DNA rearrangements of various kinds. In particular, the Holliday junction (HJ) 2 is the central intermediate in DNA recombination and repair of collapsed replication forks, whereas cruciforms or stem-loop structures are four-way junctions due to intrastrand base pairing of inverted repeats in DNA, RNA, or DNA-RNA hybrids. These structures have a regulatory role but may also pose a threat to genome stability and cause replication, transcription, and translation stall, calling for specific mechanisms for their removal. Two classes of well characterized enzymes are able to resolve HJ: resolvases, which are responsible for its cleavage and formation of crossover products (1, 2), and specialized helicases, which promote ATP hydrolysis-dependent branch migration of the junction (3-5). In archaea, representatives of the two classes of enzymes are the resolvase Hjc (6 -8) and the helicase Hjm (9), respectively. Diverse non-enzymatic architectural proteins whose action induces structural modification of DNA, and in particular DNA supercoiling, share the ability to bind four-way structures (10). For instance, the Structural Maintenance of Chromosomes (SMC) subunits of the cohesin and condensin complexes and the human DEK protein (which is involved in acute myeloid leukemias an...

“…Indeed it was shown that a lack of reverse gyrase leads to a significant reduction in the growth rate at a high temperature (13). Several functions related to maintenance of genome integrity have been proposed for reverse gyrase, including a role in rewinding of DNA strands after passage of a transcription complex or during migration of recombination junctions (15,16). Reverse gyrase is able to anneal single-stranded DNA circles, also suggesting an action as a DNA renaturase (14).…”

mentioning

confidence: 99%

“…It was also proposed that reverse gyrase could act as a "resolvase" by removing abnormal DNA structures that impede replication and transcription processes (4). Lastly, it has been shown that reverse gyrase is recruited to DNA in vivo after UV irradiation (15) and that it plays a role in preventing double-stranded DNA breakage at high temperatures (16).…”

mentioning

confidence: 99%

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

Tour

Amrani

Cossard

et al. 2008

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.The majority of DNA processing including DNA replication, transcription, repair, and recombination requires resolution of topological problems linked to the separation of the two DNA strands. Topoisomerases and helicases are two classes of enzymes specialized for these processes. Helicases catalyze disruption of hydrogen bonds between the two strands (1). Topoisomerases catalyze the interconversion of topological isomers of DNA and are required for the resolution of torsional stress and the unlinking of topologically intertwined molecules (2, 3). Several examples show that helicases and topoisomerases can act in concert (4 -6). As a model for this helicase/topoisomerase association, reverse gyrase is a unique enzyme composed of a N-terminal helicase-like domain and a C-terminal type IA topoisomerase domain, usually on a single polypeptide (7).Reverse gyrase has the almost unique property of introducing positive supercoils into a covalently closed DNA at the expense of ATP (8, 9). This enzyme is only found in archaeal and bacterial hyperthermophiles (10, 11) and is the only known hyperthermophile-specific protein (12), indicating an important role for life at high temperature. Indeed it was shown that a lack of reverse gyrase leads to a significant reduction in the growth rate at a high temperature (13). Several functions related to maintenance of genome integrity have been proposed for reverse gyrase, including a role in rewinding of DNA strands after passage of a transcription complex or during migration of recombination junctions (15, 16). Reverse gyrase is able to anneal single-stranded DNA circles, also suggesting an action as a DNA renaturase (14). It was also proposed that reverse gyrase could act as a "resolvase" by removing abnormal DNA structures that impede replication and transcription processes (4). Lastly, it has been shown that reverse gyrase is recruited to DNA in vivo after UV irradi...