RESUMO
The advance of a DNA replication fork requires an unwinding of the parental double helix. This in turn creates a positive superhelical stress, a (+)-DeltaLk, that must be relaxed by topoisomerases for replication to proceed. Surprisingly, partially replicated plasmids with a (+)-DeltaLk were not supercoiled nor were the replicated arms interwound in precatenanes. The electrophoretic mobility of these molecules indicated that they have no net writhe. Instead, the (+)-DeltaLk is absorbed by a regression of the replication fork. As the parental DNA strands re-anneal, the resultant displaced daughter strands base pair to each other to form a four-way junction at the replication fork, which is locally identical to a Holliday junction in recombination. We showed by restriction endonuclease digestion that the junction can form at either the terminus or the origin of replication and we visualized the structure with scanning force microscopy. We discuss possible physiological implications of the junction for stalled replication in vivo.
Assuntos
Replicação do DNA , DNA Bacteriano/ultraestrutura , Plasmídeos/ultraestrutura , DNA Bacteriano/metabolismo , Microscopia de Força Atômica , Modelos Genéticos , Conformação de Ácido Nucleico , Desnaturação de Ácido Nucleico , Renaturação de Ácido Nucleico , Plasmídeos/metabolismoRESUMO
We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites. For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled. Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork. We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.
Assuntos
Replicação do DNA/genética , DNA Super-Helicoidal/química , DNA Super-Helicoidal/ultraestrutura , Eletroforese em Gel de Ágar , Escherichia coli , Isomerismo , Microscopia Eletrônica , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/ultraestruturaRESUMO
Type II DNA topoisomerases catalyze the interconversion of DNA topoisomers by transporting one DNA segment through another. The steady-state fraction of knotted or catenated DNA molecules produced by prokaryotic and eukaryotic type II topoisomerases was found to be as much as 80 times lower than at thermodynamic equilibrium. These enzymes also yielded a tighter distribution of linking number topoisomers than at equilibrium. Thus, topoisomerases do not merely catalyze passage of randomly juxtaposed DNA segments but control a global property of DNA, its topology. The results imply that type II topoisomerases use the energy of adenosine triphosphate hydrolysis to preferentially remove the topological links that provide barriers to DNA segregation.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , DNA Circular/metabolismo , DNA Super-Helicoidal/metabolismo , Conformação de Ácido Nucleico , Trifosfato de Adenosina/metabolismo , Animais , DNA Topoisomerase IV , DNA Topoisomerases Tipo II/química , DNA Circular/química , DNA Super-Helicoidal/química , DNA Viral/química , DNA Viral/metabolismo , Drosophila melanogaster/enzimologia , Escherichia coli/enzimologia , Humanos , Hidrólise , Modelos Químicos , Myoviridae/enzimologia , Saccharomyces cerevisiae/enzimologia , TermodinâmicaRESUMO
DNA gyrase and topoisomerase IV (Topo IV) have distinct roles as unlinking enzymes during DNA replication despite 40% sequence identity between them. DNA gyrase unlinks replicating DNA by introducing negative supercoils while Topo IV decatenates the two daughter molecules. For this study, we measured the rates of unlinking of various topoisomers of DNA by DNA gyrase and Topo IV. Each enzyme has marked preferences for certain strand-passage reactions. DNA gyrase is a relatively poor decatenase, catalyzing strand-passage events that result in supercoiling at rates several orders of magnitude faster than those causing decatenation. Topo IV, in contrast, decatenates linked circles 10-40 times more quickly than it removes the intramolecular crossings from supercoiled DNA. Supercoiled catenanes are unlinked at an even more increased rate by Topo IV. Thus, the supercoils augment decatenation rather than compete with catenane crossings for their removal. Knot crossings and the crossings of multiply interlinked catenanes are also preferentially removed by Topo IV. This ability of Topo IV to selectively unlink catenated molecules mirrors its key role in decatenation of replicating chromosomes in vivo.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Escherichia coli/enzimologia , Adenilil Imidodifosfato/metabolismo , Autorradiografia , Replicação do DNA , DNA Topoisomerase IV , Cinética , Conformação de Ácido NucleicoRESUMO
Following a DNA strand exchange reaction, RecA protein remains bound to the hybrid DNA product. DNA strand exchange reactions were carried out under optimal conditions in the presence of both RecA protein and SSB protein. As monitored by a sensitive DNA underwinding assay, all of the RecA protein present in the RecA nucleoprotein filament that initiates the strand exchange reaction can be accounted for on the hybrid DNA. As shown elsewhere, the SSB is bound to the displaced single DNA strand. Previous studies showed that RecA protein will dissociate from dsDNA when ADP levels build up, or transfer from dsDNA to ssDNA when the latter is not bound by SSB. The present work (done with ATP regeneration and SSB) shows that efficient strand exchange occurs in the absence of a net dissociation or transfer of RecA monomers from the filament. Such a dissociation or transfer is therefore not a mechanistic requirement for DNA strand exchange. The results provide evidence against some models proposed for the DNA strand exchange mechanism.