Synthesis and dissolution of hemicatenanes by type IA DNA topoisomerases

Abstract

Type IA DNA topoisomerases work with a unique mechanism of strand passage through an enzyme-bridged, ssDNA gate, thus enabling them to carry out diverse reactions in processing structures important for replication, recombination, and repair. Here we report a unique reaction mediated by an archaeal type IA topoisomerase, the synthesis and dissolution of hemicatenanes. We cloned, purified, and characterized an unusual type IA enzyme from a hyperthermophilic archaeum, Nanoarchaeum equitans, which is split into two pieces. The recombinant heterodimeric enzyme has the expected activities in its preference of relaxing negatively supercoiled DNA. Its amino acid sequence and cleavage site sequence analysis suggest that it is topoisomerase III, and therefore we named it "NeqTop3." At high enzyme concentrations, NeqTop3 can generate high-molecular-weight DNA networks. Biochemical and electron microscopic data indicate that the DNA networks are connected through hemicatenane linkages. The hemicatenane formation likely is mediated by the singlestrand passage through denatured bubbles in the substrate DNA under high temperature. NeqTop3 at lower concentrations can reverse hemicatenanes. A complex of human topoisomerase 3α, Bloom helicase, and RecQ-mediated genome instability protein 1 and 2 can partially disentangle the hemicatenane network. Both the formation and dissolution of hemicatenanes by type IA topoisomerases demonstrate that these enzymes have an important role in regulating intermediates from replication, recombination, and repair.DNA repair | DNA topology | genetic recombination | double Holliday junction T ype IA DNA topoisomerases are ubiquitous and highly conserved enzymes that have essential functions for life. They can efficiently alleviate DNA topological stress arising from unwinding and rewinding DNA during the processes of replication, transcription, recombination, repair, and chromatin remodeling (1-4). These enzymes accomplish the feat of topological transformation via strand passage through a reversible, enzymebridged, single-strand break. The transient break is generated by a reversible transesterification reaction: The active-site tyrosine serves as a nucleophile to create phosphodiester linkage to the nascent 5′-phosphoryl end; the 3′ end is bound noncovalently to the enzyme, and its reversal can reseal the break. This unique mechanism of strand passage through a tethered DNA gate allows type IA enzymes to carry out reactions other than solely serving as a swivel to relieve topological stress. The coordinated action of type IA enzymes with DNA helicases adds further versatility as a tool in regulating the production and resolution of intermediates generated during recombination and repair (5).Type IA enzymes are present in all three domains of life, and they frequently exist in multiple forms in individual organisms. In eubacteria, there are two closely related isozymes of topoisomerase I (Top1) and III (Top3). Archaea have Top1 and/or Top3, and the hyperthermophiles have an additional type IA enzy...

DNA topoisomerase VIII: a novel subfamily of type IIB topoisomerases encoded by free or integrated plasmids in Archaea and Bacteria

“…These plasmidic topoisomerase III enzymes are used either as decatenases to help the faithful segregation of these large plasmids with low copy number, or as swivels during conjugation to facilitate the unwinding of the donor strand that is transferred to the recipient host (65). Alternatively, they may also be involved in the resolution of hemicatenane structures during plasmid replication or recombination (66). Notably, the plasmids pPPM1a of P. polymyxa and pAV109 of P. alvei encode a DNA topoisomerase III, in addition to a topoisomerase VIII.…”

Topological Behavior of Plasmid DNA

“…Historically, hemicatenanes have been observed but have been hard to study because they were hard to make. Recently, purification of the NeqTop3 enzyme from the hyperthermophilic archaeum, Nanoarchaeum equitans , has solved the problem (183). This thermophile encodes a type IA topoisomerase that works at 80°.…”

“…Convergent branch migration of a double Holliday junction can generate a hemicatenane. Reproduced from reference 183 with permission from the National Academy of Sciences. doi:10.1128/microbiolspec.PLAS-0036-2014.f16…”

Animal Mitochondrial DNA Replication

“…The X arcs observed by 2DAGE, which traditionally correspond to various cruciform structures that may result from recombination or replication termination [81, 83–85], were abundant primarily when the fragment harboring the major non-coding region (NCR) of the nematode mtDNA was probed. Treatment of the mtNA with a combination of S1 nuclease and E.coli resolvase RusA, highly specific for resolving Holiday junctions, reduced the X arc signal substantially, suggesting that a sub-fraction of the X-shaped DNA molecules are possibly hemicatenanes, a DNA species resulting from the convergence of two Holiday junctions, or replication fork stalling [86]. The rolling circle mechanism is a robust mechanism to assure efficient production of genomes as is exploited by various bacteriophages, such as Phi29, T4, λ and M13 [87–90].…”