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

Abstract

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...

Synergic and Opposing Activities of Thermophilic RecQ-like Helicase and Topoisomerase 3 Proteins in Holliday Junction Processing and Replication Fork Stabilization

“…The oligonu- cleotide A1, labeled with the Cy5 fluorophore at its 5Ј-end, was used as single-strand DNA substrate in DNA binding assay. HJ and Y-shaped fork were prepared by annealing oligonucleotides in the appropriate combinations: HJ, A1-A2-A3-A4; Y-shaped fork, A1-A2 as described previously (26). The fork regression substrate contained a 21-base oligonucleotide as the leading daughter strand and was prepared as described (32), with the difference that two oligonucleotides were labeled with different fluorophores (i.e.…”

“…Recombinant S. solfataricus SsTop3 was expressed and purified as described previously for the two reverse gyrases (30). Recombinant His-tagged E. coli topoisomerase 3 was purified as described previously (26). The RecQ protein was overexpressed in E. coli BL21-AI strain from Pet-29a-RecQ.…”

“…Reactions were terminated by adding 5ϫ stop solution (0.5% SDS, 40 mM EDTA, 0.5 mg/ml proteinase K, 20% glycerol), immediately loaded on 8% polyacrylamide gel containing 0.1% SDS and run in 0.5ϫ Tris-borate-EDTA buffer at 150 V/cm. Reaction substrates and products were analyzed as described previously (26) by gel imaging on a VersaDoc 4000 TM (Bio-Rad) using the preset laser excitation and emission setting for the Cy5 fluorophore. The relative percentage of each DNA species in each reaction was determined by Quantity One software and used to calculate the corresponding absolute amount (expressed in nanomoles).…”

“…Samples were subjected to gel electrophoresis as described for the DNA helicase assay. Reaction substrates and products were analyzed by gel imaging on a VersaDoc 4000 TM (Bio-Rad) using the preset laser excitation and emission setting for Cy5 and Cy3 fluorophores as described previously (26).…”

“…RecQ-Top3 complexes show structural and functional similarity with reverse gyrase, a peculiar enzyme specific to thermophilic archaea and bacteria, consisting of fused SF2 helicase-like and topoisomerase IA domains, which induces positive supercoiling and plays a role in genome stability in organisms living at high temperature (22)(23)(24)(25)(26). The analogies between RecQTop3 complexes and reverse gyrase led Plank and Hsieh (27) to propose the term "helicase-appended topoisomerases."…”