Dora B. Krimer

DNA knotting caused by head-on collision of transcription and replication.

Collision of transcription and replication is uncommon, but the reason for nature to avoid this type of collision is still poorly understood. In Escherichia coli pBR322 is unstable and rapidly lost without selective pressure. Stability can be rescued if transcription of the tetracycline-resistance gene (Tet(R)), progressing against replication, is avoided. We investigated the topological consequences of the collision of transcription and replication in pBR322-derived plasmids where head-on collision between the replication fork and the RNA polymerase transcribing the Tet(R) gene was allowed or avoided. The results obtained indicate that this type of collision triggers knotting of the daughter duplexes behind the fork. We propose this deleterious topological consequence could explain the instability of pBR322 and could be also one of the reasons for nature to avoid head-on collision of transcription and replication.

Transcription Termination Factor reb1p Causes Two Replication Fork Barriers at Its Cognate Sites in Fission Yeast Ribosomal DNA In Vivo

ABSTRACT Polar replication fork barriers (RFBs) near the 3′ end of the rRNA transcriptional unit are a conserved feature of ribosomal DNA (rDNA) replication in eukaryotes. In the mouse, in vivo studies indicate that the cis-acting Sal boxes required for rRNA transcription termination are also involved in replication fork blockage. On the contrary, in the budding yeast Saccharomyces cerevisiae, the rRNA transcription termination factors are not required for RFBs. Here we characterized the rDNA RFBs in the fission yeast Schizosaccharomyces pombe. S. pombe rDNA contains three closely spaced polar replication barriers named RFB1, RFB2, and RFB3 in the 3′ to 5′ order. The transcription termination protein reb1 and its two binding sites, present at the 3′ end of the coding region, were required for fork arrest at RFB2 and RFB3 in vivo. On the other hand, fork arrest at the strongest RFB1 barrier was independent of the above transcription termination factors. Therefore, RFB2 and RFB3 resemble the barriers present in the mouse rDNA, whereas RFB1 is similar to the budding yeast RFBs. These results suggest that during evolution, cis- and trans-acting factors required for rRNA transcription termination became involved in replication fork blockage also. S. pombe is suggested to be a transitional species in which both mechanisms coexist.

The Mating Type Switch-Activating Protein Sap1 Is Required for Replication Fork Arrest at the rRNA Genes of Fission Yeast

ABSTRACT Schizosaccharomyces pombe rRNA genes contain three replication fork barriers (RFB1-3) located in the nontranscribed spacer. RFB2 and RFB3 require binding of the transcription terminator factor Reb1p to two identical recognition sequences that colocalize with these barriers. RFB1, which is the strongest of the three barriers, functions in a Reb1p-independent manner, and cognate DNA-binding proteins for this barrier have not been identified yet. Here we functionally define RFB1 within a 78-bp sequence located near the 3′ end of the rRNA coding region. A protein that specifically binds to this sequence was purified by affinity chromatography and identified as Sap1p by mass spectrometry. Specific binding to RFB1 was confirmed by using Sap1p expressed in Escherichia coli. Sap1p is essential for viability and is required for efficient mating-type switching. Mutations in RFB1 that precluded formation of the Sap1p-RFB1 complex systematically abolished replication barrier function, indicating that Sap1p is required for replication fork blockage at RFB1.

Interplay of DNA supercoiling and catenation during the segregation of sister duplexes

The discrete regulation of supercoiling, catenation and knotting by DNA topoisomerases is well documented both in vivo and in vitro, but the interplay between them is still poorly understood. Here we studied DNA catenanes of bacterial plasmids arising as a result of DNA replication in Escherichia coli cells whose topoisomerase IV activity was inhibited. We combined high-resolution two-dimensional agarose gel electrophoresis with numerical simulations in order to better understand the relationship between the negative supercoiling of DNA generated by DNA gyrase and the DNA interlinking resulting from replication of circular DNA molecules. We showed that in those replication intermediates formed in vivo, catenation and negative supercoiling compete with each other. In interlinked molecules with high catenation numbers negative supercoiling is greatly limited. However, when interlinking decreases, as required for the segregation of newly replicated sister duplexes, their negative supercoiling increases. This observation indicates that negative supercoiling plays an active role during progressive decatenation of newly replicated DNA molecules in vivo.

Knotting dynamics during DNA replication

The topology of plasmid DNA changes continuously as replication progresses. But the dynamics of the process remains to be fully understood. Knotted bubbles form when topo IV knots the daughter duplexes behind the fork in response to their degree of intertwining. Here, we show that knotted bubbles can form during unimpaired DNA replication, but they become more evident in partially replicated intermediates containing a stalled fork. To learn more about the dynamics of knot formation as replication advances, we used two‐dimensional agarose gel electrophoresis to identify knotted bubbles in partially replicated molecules in which the replication fork stalled at different stages of the process. The number and complexity of knotted bubbles rose as a function of bubble size, suggesting that knotting is affected by both precatenane density and bubble size.

Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication

DNA topology plays a crucial role in all living cells. In prokaryotes, negative supercoiling is required to initiate replication and either negative or positive supercoiling assists decatenation. The role of DNA knots, however, remains a mystery. Knots are very harmful for cells if not removed efficiently, but DNA molecules become knotted in vivo. If knots are deleterious, why then does DNA become knotted? Here, we used classical genetics, high-resolution 2D agarose gel electrophoresis and atomic force microscopy to show that topoisomerase IV (Topo IV), one of the two type-II DNA topoisomerases in bacteria, is responsible for the knotting and unknotting of sister duplexes during DNA replication. We propose that when progression of the replication forks is impaired, sister duplexes become loosely intertwined. Under these conditions, Topo IV inadvertently makes the strand passages that lead to the formation of knots and removes them later on to allow their correct segregation.

Replication Fork Reversal Occurs Spontaneously after Digestion but Is Constrained in Supercoiled Domains

Replication fork reversal was investigated in undigested and linearized replication intermediates of bacterial DNA plasmids containing a stalled fork. Two-dimensional agarose gel electrophoresis, a branch migration and extrusion assay, electron microscopy, and DNA-psoralen cross-linking were used to show that extensive replication fork reversal and extrusion of the nascent-nascent duplex occurs spontaneously after DNA nicking and restriction enzyme digestion but that fork retreat is severely limited in covalently closed supercoiled domains.

A computer model for the analysis of DNA replication intermediates by two-dimensional agarose gel electrophoresis

We present a computer model to predict the patterns expected for the replication intermediates (RIs) of DNA fragments analyzed by neutral/neutral two-dimensional (2D) agarose gel electrophoresis. The model relies on the mode of replication (uni- or bi-directional), the electrophoretic mobility of linear DNA fragments and the retardation caused by the three-dimensional shape of non-linear molecules. The utility of this model is demonstrated with two examples: replication analysis of the plasmids pBR322 and pHH5.8 in Escherichia coli after digestions with EcoRI and HindIII, respectively.

Plasmid DNA replication and topology as visualized by two-dimensional agarose gel electrophoresis

During the last 20 years, two-dimensional agarose gel electrophoresis combined with other techniques such as Polymerase Chain Reaction, helicase assay and electron microscopy, helped to characterize plasmid DNA replication and topology. Here we describe some of the most important findings that were made using this method including the characterization of uni-directional replication, replication origin interference, DNA breakage at the forks, replication fork blockage, replication knotting, replication fork reversal, the interplay of supercoiling and catenation and other changes in DNA topology that take place as replication progresses.

The fission yeast rDNA-binding protein Reb1 regulates G1 phase under nutritional stress

Yeast Reb1 and its mammalian ortholog TTF1 are conserved Myb-type DNA-binding proteins that bind to specific sites near the 3′-end of rRNA genes (rDNA). Here, they participate in the termination of transcription driven by RNA polymerase I and block DNA replication forks approaching in the opposite direction. We found that Schizosaccharomyces pombe Reb1 also upregulates transcription of the ste9+ gene that is required for nitrogen-starvation-induced growth arrest with a G1 DNA content and sexual differentiation. Ste9 activates the anaphase-promoting complex or cyclosome (‘APC/C’) in G1, targeting B-cyclin for proteasomal degradation in response to nutritional stress. Reb1 binds in vivo and in vitro to a specific DNA sequence at the promoter of ste9+, similar to the sequence recognized in the rDNA, and this binding is required for ste9+ transcriptional activation and G1 arrest. This suggests that Reb1 acts as a link between rDNA metabolism and cell cycle control in response to nutritional stress. In agreement with this new role for Reb1 in the regulation of the G1–S transition, reb1Δ and wee1ts mutations are synthetically lethal owing to the inability of these cells to lengthen G1 before entering S phase. Similarly, reb1Δ cdc10ts cells are unable to arrest in G1 and die at the semi-permissive temperature.