The mechanism of type IA topoisomerases.

The topology of cellular DNA is carefully controlled by enzymes called topoisomerases. By using single-molecule techniques, we monitored the activity of two type IA topoisomerases in real time under conditions in which single relaxation events were detected. The strict one-at-a-time removal of supercoils we observed establishes that these enzymes use an enzyme-bridged strand-passage mechanism that is well suited to their physiological roles and demonstrates a mechanistic unity with type II topoisomerases.

Topological challenges to DNA replication: conformations at the fork.

The unwinding of the parental DNA duplex during replication causes a positive linking number difference, or superhelical strain, to build up around the elongating replication fork. The branching at the fork and this strain bring about different conformations from that of (-) supercoiled DNA that is not being replicated. The replicating DNA can form (+) precatenanes, in which the daughter DNAs are intertwined, and (+) supercoils. Topoisomerases have the essential role of relieving the superhelical strain by removing these structures. Stalled replication forks of molecules with a (+) superhelical strain have the additional option of regressing, forming a four-way junction at the replication fork. This four-way junction can be acted on by recombination enzymes to restart replication. Replication and chromosome folding are made easier by topological domain barriers, which sequester the substrates for topoisomerases into defined and concentrated regions. Domain barriers also allow replicated DNA to be (-) supercoiled. We discuss the importance of replicating DNA conformations and the roles of topoisomerases, focusing on recent work from our laboratory.

Mechanism of topology simplification by type II DNA topoisomerases.

Type II DNA topoisomerases actively reduce the fractions of knotted and catenated circular DNA below thermodynamic equilibrium values. To explain this surprising finding, we designed a model in which topoisomerases introduce a sharp bend in DNA. Because the enzymes have a specific orientation relative to the bend, they act like Maxwell's demon, providing unidirectional strand passage. Quantitative analysis of the model by computer simulations proved that it can explain much of the experimental data. The required sharp DNA bend was demonstrated by a greatly increased cyclization of short DNA fragments from topoisomerase binding and by direct visualization with electron microscopy.

Positive torsional strain causes the formation of a four-way junction at replication forks.

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.

Bypass of heterology during strand transfer by Saccharomyces cerevisiae Rad51 protein.

During recombination-mediated repair of DNA double-strand breaks, strand transfer proteins must distinguish a homologous repair template from closely related genomic sequences. However, some tolerance by strand transfer proteins for sequence differences is also critical: too much stringency will prevent recombination between different alleles of the same gene, but too much tolerance will lead to illegitimate recombination. We characterized the heterology tolerance of Saccharomyces cerevisiae Rad51 by testing bypass of small heterologous inserts in either the single- or double-stranded substrate of an in vitro strand transfer reaction that models the early steps of homologous recombination. We found that the yeast protein is rather stringent, only tolerating heterologies up to 9 bases long. The efficiency of heterology bypass depends on whether the insert is in the single- or double-stranded substrate, as well as on the location of the insert relative to the end of the double-stranded linear substrate. Rad51 is distinct in that it can catalyze strand transfer in either the 3'-->5' or 5'-->3' direction. We found that bypass of heterology was independent of the polarity of strand transfer, suggesting that the mechanism of 5'-->3' transfer is the same as that of 3'-->5' transfer.

Preferential relaxation of positively supercoiled DNA by E. coli topoisomerase IV in single-molecule and ensemble measurements.

We show that positively supercoiled [(+) SC] DNA is the preferred substrate for Escherichia coli topoisomerase IV (topo IV). We measured topo IV relaxation of (-) and (+) supercoils in real time on single, tethered DNA molecules to complement ensemble experiments. We find that the preference for (+) SC DNA is complete at low enzyme concentration. Otherwise, topo IV relaxed (+) supercoils at a 20-fold faster rate than (-) supercoils, due primarily to about a 10-fold increase in processivity with (+) SC DNA. The preferential cleavage of (+) SC DNA in a competition experiment showed that substrate discrimination can take place prior to strand passage in the presence or absence of ATP. We propose that topo IV discriminates between (-) and (+) supercoiled DNA by recognition of the geometry of (+) SC DNA. Our results explain how topo IV can rapidly remove (+) supercoils to support DNA replication without relaxing the essential (-) supercoils of the chromosome. They also show that the rate of supercoil relaxation by topo IV is several orders of magnitude faster than hitherto appreciated, so that a single enzyme may suffice at each replication fork.

DNA microarray analysis of gene expression in response to physiological and genetic changes that affect tryptophan metabolism in Escherichia coli.

We investigated the global changes in mRNA abundance in Escherichia coli elicited by various perturbations of tryptophan metabolism. To do so we printed DNA microarrays containing 95% of all annotated E. coli ORFs. We determined the expression profile that is predominantly dictated by the activity of the tryptophan repressor. Only three operons, trp, mtr, and aroH, exhibited appreciable expression changes consistent with this profile. The quantitative changes we observed in mRNA levels for the five genes of the trp operon were consistent within a factor of 2, with expectations based on established Trp protein levels. Several operons known to be regulated by the TyrR protein, aroF-tyrA, aroL, aroP, and aroG, were down-regulated on addition of tryptophan. TyrR can be activated by any one of the three aromatic amino acids. Only one operon, tnaAB, was significantly activated by the presence of tryptophan in the medium. We uncovered a plethora of likely indirect effects of changes in tryptophan metabolism on intracellular mRNA pools, most prominent of which was the sensitivity of arginine biosynthetic operons to tryptophan starvation.

Analysis of topoisomerase function in bacterial replication fork movement: use of DNA microarrays.

We used DNA microarrays of the Escherichia coli genome to trace the progression of chromosomal replication forks in synchronized cells. We found that both DNA gyrase and topoisomerase IV (topo IV) promote replication fork progression. When both enzymes were inhibited, the replication fork stopped rapidly. The elongation rate with topo IV alone was 1/3 of normal. Genetic data confirmed and extended these results. Inactivation of gyrase alone caused a slow stop of replication. Topo IV activity was sufficient to prevent accumulation of (+) supercoils in plasmid DNA in vivo, suggesting that topo IV can promote replication by removing (+) supercoils in front of the chromosomal fork.

Roles of topoisomerases in maintaining steady-state DNA supercoiling in Escherichia coli.

DNA supercoiling is essential for bacterial cell survival. We demonstrated that DNA topoisomerase IV, acting in concert with topoisomerase I and gyrase, makes an important contribution to the steady-state level of supercoiling in Escherichia coli. Following inhibition of gyrase, topoisomerase IV alone relaxed plasmid DNA to a final supercoiling density (sigma) of -0.015 at an initial rate of 0.8 links min(-1). Topoisomerase I relaxed DNA at a faster rate, 5 links min(-1), but only to a sigma of -0.05. Inhibition of topoisomerase IV in wild-type cells increased supercoiling to approximately the same level as in a mutant lacking topoisomerase I activity (to sigma = -0.08). The role of topoisomerase IV was revealed by two functional assays. Removal of both topoisomerase I and topoisomerase IV caused the DNA to become hyper-negatively supercoiled (sigma = -0.09), greatly stimulating transcription from the supercoiling sensitive leu-500 promoter and increasing the number of supercoils trapped by lambda integrase site-specific recombination.

Equilibrium distributions of topological states in circular DNA: interplay of supercoiling and knotting.

Two variables define the topological state of closed double-stranded DNA: the knot type, K, and DeltaLk, the linking number difference from relaxed DNA. The equilibrium distribution of probabilities of these states, P(DeltaLk, K), is related to two conditional distributions: P(DeltaLk|K), the distribution of DeltaLk for a particular K, and P(K|DeltaLk) and also to two simple distributions: P(DeltaLk), the distribution of DeltaLk irrespective of K, and P(K). We explored the relationships between these distributions. P(DeltaLk, K), P(DeltaLk), and P(K|DeltaLk) were calculated from the simulated distributions of P(DeltaLk|K) and of P(K). The calculated distributions agreed with previous experimental and theoretical results and greatly advanced on them. Our major focus was on P(K|DeltaLk), the distribution of knot types for a particular value of DeltaLk, which had not been evaluated previously. We found that unknotted circular DNA is not the most probable state beyond small values of DeltaLk. Highly chiral knotted DNA has a lower free energy because it has less torsional deformation. Surprisingly, even at |DeltaLk| > 12, only one or two knot types dominate the P(K|DeltaLk) distribution despite the huge number of knots of comparable complexity. A large fraction of the knots found belong to the small family of torus knots. The relationship between supercoiling and knotting in vivo is discussed.

Knot what we thought before: the twisted story of replication.

DNA replication requires the unwinding of the parental duplex, which generates (+) supercoiling ahead of the replication fork. It has been thought that removal of these (+) supercoils was the only method of unlinking the parental strands. Recent evidence implies that supercoils can diffuse across the replication fork, resulting in interwound replicated strands called precatenanes. Topoisomerases can then act both in front of and behind the replication fork. A new study by Sogo et al. [J Mol Biol 1999;286:637-643 (Ref. 1)], using a topological analysis, provides the best evidence that precatenanes exist in negatively supercoiled, partially replicated molecules in vivo.

13S condensin actively reconfigures DNA by introducing global positive writhe: implications for chromosome condensation.

Xenopus 13S condensin converts interphase chromatin into mitotic-like chromosomes, and, in the presence of ATP and a type I topoisomerase, introduces (+) supercoils into DNA. The specific production of (+) trefoil knots in the presence of condensin and a type II topoisomerase shows that condensin reconfigures DNA by introducing an ordered, global, (+) writhe. Knotting required ATP hydrolysis and cell cycle-specific phosphorylation of condensin. Condensin bound preferentially to (+) supercoiled DNA in the presence of ATP but not in its absence. Our results suggest a mechanism for the compaction of chromatin by condensin during mitosis.

The topological mechanism of phage lambda integrase.

Bacteriophage lambda integrase (Int) is a versatile site-specific recombinase. In concert with other proteins, it mediates phage integration into and excision out of the bacterial chromosome. Int recombines intramolecular sites in inverse or direct orientation or sites on separate DNA molecules. This wide spectrum of Int-mediated reactions has, however, hindered our understanding of the topology of Int recombination. By systematically analyzing the topology of Int reaction products and using a mathematical method called tangles, we deduce a unified model for Int recombination. We find that, even in the absence of (-) supercoiling, all Int reactions are chiral, producing one of two possible enantiomers of each product. We propose that this chirality reflects a right-handed DNA crossing within or between recombination sites in the synaptic complex that favors formation of right-handed Holliday junction intermediates. We demonstrate that the change in linking number associated with excisive inversion with relaxed DNA is equally +2 and -2, reflecting two different substrates with different topology but the same chirality. Additionally, we deduce that integrative Int recombination differs from excisive recombination only by additional plectonemic (-) DNA crossings in the synaptic complex: two with supercoiled substrates and one with relaxed substrates. The generality of our results is indicated by our finding that two other members of the integrase superfamily of recombinases, Flp of yeast and Cre of phage P1, show the same intrinsic chirality as lambda Int.