Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes

Topological constraints impair RNA polymerase II transcription and causes instability of plasmid-borne convergent genes Marı ´a L. Garcı ´a-Rubio and Andre ´s Aguilera* Centro Andaluz de Biologı ´a Molecular y Medicina Regenerativa CABIMER, Universidad de Sevilla-CSIC, Av. Ame ´rico Vespucio s/n 41092 Sevilla, and Departamento de Gene ´tica, Universidad de Sevilla, 41012 Sevilla, Spain Received June 30, 2011; Revised September 8, 2011; Accepted September 21, 2011 ABSTRACT Despite the theoretical bases for the association of topoisomerases and supercoiling changes with transcription and replication, our knowledge of the impact of topological constraints on transcription and replication is incomplete. Although mutation of topoisomerases affects expression and stability of the rDNA region it is not clear whether the same is the case for RNAPII transcription and genome integrity in other regions. We developed new assays in which two convergent RNAPII-driven genes are transcribed simultaneously. Plasmid-based systems were constructed with and without a transcription terminator between the two convergent transcription units, so that the impact of transcription interference could also be evaluated. Using these assays we show that Topos I and II play roles in RNAPII transcription in vivo and reduce the stability of RNAPII-transcribed genes in Saccharomyces cerevisiae. Supercoiling accumulation in convergent transcription units impairs RNAPII transcription in top1Dstrains, but Topo II is also required for efficient transcription independent of Topo I and of detectable supercoiling accumulation. Our work shows that topological constraints negatively affect RNAPII transcription and genetic integrity, and provides an assay to study gene regulation by transcription interference. INTRODUCTION DNA in its natural form in the cell is supercoiled. In eukaryotes this state facilitates its organization into chromatin around nucleosomes. DNA topoisomerases control supercoiling and their action is required in replication and transcription as well as in chromosome condensation and segregation (1,2). There are two types of DNA topoisomerases (Topo), I and II, which differ in their catalytic way of action, the first relying on a single-strand DNA cleavage, the second on a double-strand break (DSB). Topoisomerases I and II are the most representative of each type in all organisms. They are functionally interchangeable in eukaryotes, but not in bacteria. During replication DNA topoisomerases can act at different stages. In Escherichia coli reconstituted replication systems require DNA supercoiling for replication initiation and the action of DNA topoisomerases (3). However, it is during elongation of replication that topoisomerases are expected to have a key role. They are required to remove the positive supercoiling that are generated by the continuous opening of the template strands. The intertwined daughter DNA molecules resulting after replication completion require the action of type II topoisomerases for their segregation. This late function in replication cannot be performed by type I topoisomerases but only by type II (4,5). In contrast to replication, transcription does not involve a continuous separation of DNA strands. However, RNA polymerase progression through the DNA template would lead to the accumulation of local positive supercoiling ahead of the RNA polymerase and negative supercoiling behind. The twin-supercoiled-domain model was proposed to explain removal of this local supercoiling accumulation by topoisomerases during transcription (6). Evidence for transcriptional supercoiling associated with transcription has been provided in E. coli (6,7) and yeast (8,10). The relaxation of transcription-dependent supercoiling is generally considered to be the main function of Topo I, which is considered to play a swivel role in transcription (1,2). In eukaryotes such as the yeast Saccharomyces cerevisiae, the fact that top1Dmutants are viable suggests that Topo II can replace the function of Topo I; whereas the opposite is not the case, since Topo I *To whom correspondence should be addressed. Tel: +34 954 468 372; Fax: +34 954 461 664; Email: [email protected] 1050–1064 Nucleic Acids Research, 2012, Vol. 40, No. 3 Published online 13 October 2011 doi:10.1093/nar/gkr840 ßThe Author(s) 2011. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. cannot replace Topo II at the end of replication during segregation. Despite the theoretical bases for the association of topoisomerases and supercoiling changes with transcription, experimental evidence for the impact of topoisomerases deficiency and topological constraints in transcription is incomplete. In bacteria, the importance of Topo I has been well documented in rDNA transcription (11) or transcription from operons involved in amino acid metabolism (12,13). In yeast, an effect of topoisomerase activity has been reported on transcription initiation. This may be related to the need of negatively supercoiled DNA at promoter regions to facilitate transcription initiation (14). The effect of DNA topoisomerases on transcription elongation has been established for rDNA, in which the rate of RNAPI transcription is reduced when both DNA topoisomerases are inactivated in yeast (8,15). Instead, none or a poor effect has been reported for mRNA transcription as tested in endogenous and lacZ reporters fused to GAL promoters in vivo (10,16). In addition to the effect of Topo mutations in cell growth, transcription and replication, they have an important impact on genome integrity. Stability of tandem rDNA repeats in yeast has been shown to be strongly dependent on DNA topoisomeases I and II, whereas this was not the case for direct repeats outside of the rDNA region (17). Hyper-recombination has been shown in plasmid-borne direct-repeats in double top1 top2 mutants (18). It has been proposed that this is mainly the result of the failure to relax the negative rather than the positive supercoiling of DNA (18). Nevertheless, impairment of replication fork progression by obstacles or DNA lesions can lead to replication fork collapse that in turn could trigger recombination as a mechanism of replication restart (19). Thus, hyper-recombination of yeast topoisomerase mutants could be linked to their role in replication, but the functional relevance of Topo I in rDNA transcription and its large accumulation in the nucleolus, makes plausible that hyper-recombination could also be linked to transcription. Indeed, rDNA genes contain the RNAPI transcription-dependent HOT1 recombination hotspots (20,21), and one key function of the Fob1 barrier to DNA replication is to avoid replication–transcription collisions that could potentially compromise genome integrity (22). In this sense, one intriguing class of hyperrecombination mutations in yeast is composed of those of the THO complex. THO is a conserved protein complex composed of stoichiometric amounts of Hpr1, Tho2, Mft1 and Thp2, with a role in transcription elongation, mRNP biogenesis and RNA export (23). THO mutants, as well as mutants related to mRNP biogenesis factors such as the THSC/TREX-2 complex, cause a strong hyper-recombination between direct repeats that are transcription-dependent. This phenotype is linked to the co-transcriptional formation of R-loops that can impair the progression of replication (24,25). Interestingly, in E. coli mutants lacking topoisomerase I, an excess of negative supercoiling during transcription can lead to R-loops (26). Furthermore, THO and THSC mutants show synthetic growth defects in combination with top1 and top2 mutations (27). All this evidence raises the question of whether hyper-recombination in the rDNA region in topoisomerases mutants could be linked to transcription. Thus, although topological constraints and topoisomerase mutations have a clear impact in expression and stability of the rDNA locus this is not that clear for RNAPII transcription and genome integrity at regions others than rDNA. To investigate this, new and highly sensitive in vivo assays were developed based on plasmid-borne DNA sequences of increasing lengths that are transcribed form strong promoters with or without convergent transcription coming from a second promoter. We show that both top1 and top2 mutations drastically reduce RNAPII transcription, a defect that is strengthened as the length of the transcribed DNA sequence increases and by convergent transcription. Importantly, top2-1 mutation causes a significant increase in direct-repeat recombination in atop1Dmutant background; however, this hyperrecombination was predominantly independent of transcription of the direct-repeat system itself. Therefore, this study provides new evidence for the in vivo relevance of Topos I and II in RNAPII transcription and genome integrity. We propose that DNA topoisomerases has a non-interchangeable role in the maintenance of integrity of RNAPII genes providing evidence for the negative effect of topological constraints in transcription and genome stability in DNA regions beyond the rDNA. Although a similar study would need to be performed in chromosome-borne constructs, we believe that these results may also be valid for chromosomal regions, provided that local supercoiling dynamics follow universal rules. MATERIALS AND METHODS Strains and plasmids Yeast strains and plasmids are described in Supplementary Tables S1 and S2, respectively. Chromatin immunoprecipitation analysis For ChIP experiments, strains were grown in synthetic medium (SC) 2% glycerol-2% lactate to an OD 660 of 0.5 (for GAL1). For GAL1 ChIPs the culture was split in two, and one half was supplemented with 2% glucose (repressed transcription) and the other with 2% galactose (activated transcription). For P tet -based systems, yeast strains were grown in synthetic medium (SC), the culture was split in two, and one half was supplemented with doxycycline. Samples were then taken after 4 h of induction and ChIP assays were performed as described (28). Polyclonal anti-PolII (N-20) (Santa Cruz Biotechnology) and Protein A–Sepharose were used for RNAPII immunoprecipitation. The GFX purification system (Amersham) was used for the last DNA purification step. We used the PCR of the intergenic region at positions 9716–9863 of chromosome V as a negative control. Real-time quantitative PCR and calculations of the relative abundance of each DNA fragment was performed as described (29). For each experiment, the DNA ratios in the different regions were calculated from the DNA amount of these regions relative to the intergenic region. Nucleic Acids Research, 2012, Vol. 40, No. 3 1051 Median and Standard Deviation (SD) of three independent experiments are shown. Nucleic acid isolation For DNA isolation, pellets were re-suspended to a final concentration of 1.5–2 10 9 /ml in NIB pH 7.2 (17% glycerol, 50 mM MOPS, 150 mM KAc, 2 mM MgCl, 0.5 mM spermidine and 0.15 mM spermine) with 3 mg/ml Zymoliase-20T and incubated at room temperature for 25 min. Cellular suspensions were diluted seven times with chilled water and centrifuged to collect nuclei. Pellets were re-suspended in 720 ml of TE (50 mM Tris–HCl, pH 8, 20 mM EDTA) and mixed with 80 mlof 10% SDS. After phenol–chloroform–isoamylalcohol (25:24:1) extraction and ethanol precipitation, DNA was finally re-suspend in 20–30 ml of TE. Chloroquine gel electrophoresis Electrophoresis was performed at room temperature on horizontal slab gels (0.7% agarose) containing TPE buffer (50 mM Tris–phosphate [pH 7.2], 1 mM EDTA and 25 mM phosphoric acid). Chloroquine (Sigma) was added to the melted agarose from a 10 mg/ml stock prior to casting the gel and was present in the electrophoresis buffer. The buffer was re-circulated continuously during electrophoresis. The concentration of chloroquine was 4mg/ml. Each lane had 20 mg of total nucleic acids. The gels were run at 40 V for 48 h. Miscellaneous Yeast methods, a- 32 P-labeled DNA probes, northern and Southern analyses were performed according to standard procedures. RNA was isolated from mid-log phase cells. Recombination frequencies were obtained by fluctuation tests as the median value of six independent colonies isolated from SC plates. The final frequency given for each strain and condition is the mean and SD of three to four median values as described (30). Recombinants Leu+were selected in SC- Leu plates. RESULTS Convergent transcription in systems with or without terminator sequences For a fine assessment of the possible effect of topological constraints in transcription, a plasmid-based system was constructed in which a 4-kb sequence was transcribed from two convergent and differentially regulated promoters located on each side. For this construction, we fused a 2-kb lacZ fragment under the GAL1 promoter to a 2-kb lys2 fragment under the quimeric tet promoter so that transcription driven from the two promoters was convergent (Figure 1). Two versions of this system were constructed in monocopy CEN-based plasmids: one with a 0.4-kb CYC1 terminator between the lys2 and lacZ sequences (pTGlylaTER) and the other without transcription terminator (pTGlyla). The CYC1 terminator sequence contains two converging terminators so that it acts in both orientations. As expected, whereas in the pTGlyla system without terminator the 4-kb lys2-lacZ mRNA was produced when cells were cultivated either with 2% galactose (GAL1 promoter activated) or without doxycycline (tet promoter activated), but not in 2% glucose + doxycycline (GAL1 and tet promoters repressed) (Figure 1A). To confirm that the band detected in 2% galactose was driven from the GAL1 promoter and the band detected in the absence of doxycycline was driven from the tet promoter northern analysis was performed in which hybridization was made with specific ssDNA probes. As expected, the 4-kb mRNA band was specific to a DNA strand. It was detected by the Watson-chain-specific (W) probe in the absence of doxycycline (P tet active), and with the Crick-chain-specific (C) probe in galactose (P GAL1 active), according to the promoter from which each transcript was transcribed (Figure 1B). Instead, in the pTGlylaTER system either the 2-kb lacZ or the 2-kb lys2 mRNAs were detected in galactose regardless of the presence of doxycycline, or in doxycycline regardless of the carbon source, respectively (Figure 1C and D). Importantly, in the pTGlyla system, the level of 4-kb lys2-lacZ transcripts was lower when both the P GAL1 and P tet promoters were simultaneously active than when only one was active, as observed with either the lys2 or the lacZ probe (Figure 1A and B). This result is consistent with transcription interference either at the promoter or transcription stalling provided by RNAPII collisions. Since the 4-kb transcript driven from either the tet or GAL1 promoters reaches the downstream converging promoter, the latter may undergo transcription interference. When the transcripts were analyzed with specific ssDNA probes it was found that after galactose activation the P tet -driven 4-kb transcript, as detected with the W probe, was reduced, whereas the P GAL1 -driven transcript was below detection levels (Figure 1B). This indicates that since the P tet -driven RNAPII reaches P GAL1 , it causes promoter interference impeding P GAL1 activation, even though transcriptional stalling cannot be excluded either. Similarly, P GAL1 -driven transcription seems to reduce the efficiency of P tet -driven transcription, although to a lesser extent. To assay whether the northern results could be interpreted in terms of RNAPII progression, a chromatin– immunoprecipitation (ChIP) analysis was performed with anti-Rpb1 N-ter antibodies in both the pTGlylaTER and pTGlyla systems under all possible conditions (with either none, only one or both promoters activated). As can be seen in Figure 2, RNAPII recruitment was not observed in either of the regions analyzed when both promoters were repressed in either system, as expected. In the pTGlylaTER system RNAPII was detected in lys2 when only P tet was active and in both lys2 and lacZ when only P GAL1 was active (Figure 2A). The latter was consistent with transcription termination read-through observed for transcription initiated at the strong P GAL1 promoter, as observed by northern (Figure 1). In the pTGlyla system, RNAPII recruitment was observed at both the lys2 and lacZ regions when either P tet or P GAL1 were the only active promoters, whereas recruitment to one promoter was only detected when transcription was driven from the opposite promoter (Figure 2B), implying that such polymerases corresponded to elongating RNAPIIs. When both promoters 1052 Nucleic Acids Research, 2012, Vol. 40, No. 3 were active, the lys2 and lacZ signals were detected, as expected, in the pTGlylaTER system, whereas only the lys2 signal was detected in pTGlyla. This confirms that the northern analysis used can be interpreted in terms of RNAPII capacity to transcribe the different regions analyzed. Transcription elongation is sensitive to Topos I and II inactivation Next, the two constructs were used to analyze transcription in the presence of doxycycline, so that the P tet promoter was inactivated and transcription was only driven from P GAL1 . Under these conditions kinetics experiments were performed of P GAL1 -driven transcription with 2% galactose to determine the efficiency of transcription of the 4-kb lacZ-lys2 transcript in pTGlyla and the 2-kb LacZ transcript in pTGlylaTER. As can be seen in Figure 3, transcription of LacZ was clearly reduced in top1Dmutants, more reduced in top2-1 mutants and further in the double top1Dtop2-1 mutants. Transcription of the 4-kb lys2-lacZ was dramatically decreased in all single and double top mutants. This result is consistent with a defect in transcription elongation so that the longer the RNA is transcribed the stronger the effect. The double mutant showed the strongest effect and a similar dramatic decrease in the accumulation of both of the 2- and 4-kb transcripts. Different studies have revealed that yeast mutations impairing transcription elongation causes a reduction in mRNA accumulation that in most cases increases with the length of the transcript unit analyzed (23). Using plasmid-borne P GAL1 ::PHO5 and P GAL1 ::lacZ constructs containing the 1.2-kb and 3.1-kb yeast PHO5 and bacterial lacZ ORFs, respectively, under the same GAL1 promoter, the negative effect of topoisomerase mutations was confirmed in transcription of long DNA sequences. The single and double Topo mutants have a significant effect on the efficiency of transcription of the PHO5 ORF, the reduction in the accumulation of the lacZ mRNA being stronger in the single top2-1 and in the double thp1Dtop2-1 (Supplementary Figure S1). Given that transcription in both constructs is driven from the same GAL1 promoter, this result excludes the possibility Figure 1. Transcription analyses of the lys2-CYCt-lacZ and lys2-lacZ fusion constructs. (A) Northern analyses of lys2-lacZ-containing mRNAs driven from the tet and GAL1 promoters. The 3-kb XbaI-EcoRV lacZ and a 589-bp 25 S rDNA internal fragments obtained by PCR (rRNA) were used as DNA probes. (B) Northern analyses of lys2-lacZ. The oligos C and W were used as single-stranded DNA probes. (C), (D) Northern analyses of pTGlylaT-1 and pTGlylaT-5 plasmids. Arrows refers to the mRNA being produced from the specified promoter. No arrow indicates that there is no transcription from that promoter. All experiments are made in the absence of doxycycline, so that the tet promoter is permanently active. A total of 2% glucose (Glu) or galactose (Gal) are used to either repress or activate the GAL1 promoter. Other details as described in ‘Materials and Methods’ section. Nucleic Acids Research, 2012, Vol. 40, No. 3 1053 that the major transcription defects observed in top mutants occur at transcription initiation, but at elongation. In addition, consistent with the idea that eukaryotic Topos I and II can partially replace each other, we showed that overexpression of Top1 can partially suppress the lacZ mRNA accumulation defect of top2-1 mutants (Supplementary Figure S2). Convergent transcription is impaired under Topos I and II inactivation as a function of supercoiling accumulation Next, we assayed whether or not convergent transcription affected transcription efficiency. This analysis of convergent transcription was made in the absence of doxycycline to allow continuous P tet -driven transcription. Time-course kinetics was performed for the P GAL1 -driven transcripts after galactose addition. As can be seen in Figure 4, in the pTGlylaTER system with the transcription terminator the overall levels of the P tet -driven lys2 transcript did not show a significant change after activation of the converging P GAL1 . At 200 min of P GAL1 activation the overall levels of lys2 transcripts were similar to the levels before activation. At intermediate times some differences between wild-type and mutant strains were evident, but these were minor. This may be a response to cell adaptation to new conditions. Importantly, at 200 min of activation the lys2 transcript levels were the same in wild-type and mutant strains, with a slight decrease in top1Dtop2-1 mutants. Instead, transcription from the GAL1 promoter, as determined by accumulation of the 2-kb lacZ transcript, was clearly affected in top1Dand top2-1 mutants (38–40% of wild-type levels) and much more in the double top1D top2-1 mutants (10% of wild-type levels) (Figure 4A). The clear impairment in transcription of the 2-kb lacZ transcript caused by topoisomerase mutations as compared to the poor or no effect on the 2-kb lys2 transcript (both transcripts are of the same size but are driven from promoters of different strengths), indicates that the higher the transcription rate the higher the requirement of topoisomerases. In the pTGlyla system there is little change in the total amount of 4-kb transcripts (Figure 4B) during the kinetics performed. As shown above, these results indicate that pTGlyla, which contains no terminator sequence between the converging promoters, undergoes promoter interference caused at P GAL1 by the RNAPII coming from P tet . However, despite the interference phenomenon, transcripts were moderately low in single top1 and top2 mutants (60–65% of wild-type levels after 200 min activation) and lower in double top1 top2 mutants (38–45%) as compared to wild-type levels. This is consistent with the importance of topoisomerase activities in converging transcription. It is worth noting the different topoisomerase requirements of transcription of the 4-kb lys2-lacZ and 2-kb lys2 fragments in the pTGlyla and pTGlylaTER systems (compare Figures 4A. up and D, bottom). This is due to the longer length of the lacZ-lys2 fragment, consistent with the conclusion that the longer the transcript the higher the dependency of transcription on topoisomerase activity (Figure 1). Altogether, the results indicate that topoisomerases I and II are required for efficient RNAPII transcription elongation. To assay whether the transcription defect caused by top mutations was linked to the incapacity to resolve Figure 2. Distribution of RNAPII along the lys2-lacZ transcription units of plasmids pTGlyla and pTGlylaTER. ChIP analyses in the wild-type strain (HRN1-4A) carrying the plasmids pTGlylaTER (A) or TGlyla (B). Schemes of the gene and the PCR-amplified fragments are shown. DNA ratios in regions 1–4 were calculated from the amounts obtained for these regions relative to the amounts of the intergenic region. ChIPs were performed from three independent cultures, and quantitative PCRs were repeated three times for each culture. SDs are indicated as error bars. 1054 Nucleic Acids Research, 2012, Vol. 40, No. 3 supercoiling, rather than to a specific function of any of the topoisomerases at specific target DNA sites, supercoiling of the plasmids was analyzed carrying both the pTGlylaTER and pTGlyla systems under conditions of transcription activation of both promoters. As can be seen in Figure 5, a large proportion of supercoiled DNA is accumulated in top mutants, in particular in top1 and top1 top2 when both promoters are active, but not when they were inactive. Experiments were performed similarly to previous reports using chloroquine concentrations in which all topoisomers show negative supercoiling (9). Importantly, accumulation of the supercoiled plasmid band is clearly stronger in pTGlyla versus pTGlylaTER, consistent with the conclusion that the longer the length of the transcription unit, the higher the change in supercoiling produced by transcription and the stronger the requirement of topoisomerases. Even though on a theoretical basis the impact of transcription on DNA supercoiling is local, TOP1 inactivation has a global impact on plasmid supercoiling as has been previously reported (9). As previously shown (9) accumulation of global supercoiling was not evident in top2-1 mutants. Efficient RNAPII-driven convergent transcription requires Topos I and II So far, all our previous experiments were performed at 30C, a semi-permissive temperature to allow growth of Figure 3. Transcription analyses of lys2-CYCt-lacZ and lys2-lacZ fusion transcript driven form the GAL1 promoter. (A) Northern analyses of lys2-CYCt-lacZ containing mRNAs driven from the GAL1 promoter in wild-type (HRN1-4A), top1D(TOHR-11A), top2-1 (TORH-6A) and top1D top2-1 (TOHR-13C) strains. Mid-log phase plasmid-transformed cells were diluted in 3% glycerol–2%lactate synthetic complete (SC)-Trp medium plus doxycycline and diluted into identical fresh media to an OD 600 of 0.4 and incubated for 16 h. Galactose was then added and samples were taken for northern analyses at different times. RNA levels in arbitrary units were obtained by quantification of signals intensities in a FUJI FLA 5000 and normalized with respect to rRNA levels of each sample. Wild-type mRNA levels were taken as 100%. (B) Northern analyses of lys-lacZ-containing mRNAs driven from the GAL1 promoter using the same strains as in A. Nucleic Acids Research, 2012, Vol. 40, No. 3 1055 Figure 4. Northern analyses of convergent transcription in the lys2-CYCt-lacZ and lys2-lacZ fusion constructs. (A) Northern analyses of lys2-CYCt-lacZ mRNAs driven from the tet and GAL1 promoters in wild-type (HRN1-4A), top1D(TOHR-11A), top2-1 (TORH-6A) and top1D top2-1 (TOHR-13C) strains. The 3-Kb XbaI-EcoRV lacZ internal fragment were used as DNA probe. (B) Northern analyses of lys2-lacZ mRNAs driven from the P tet and P GAL1 promoters. Other details as described for Figure 3. 1056 Nucleic Acids Research, 2012, Vol. 40, No. 3 top2-1 thermosensitive mutants. Yet, it was possible to see a synergistic effect on double top1 top2 mutants suggesting that Topo II has a role in transcription in vivo. This conclusion was confirmed by assaying the effect on complete Top II inactivation. These experiments were repeated on convergent transcription in both the pTGlyla and pTGlylaTER systems after shifting cells to the restrictive temperature of 37C. As can be seen in Figure 6A, in the pTGlylaTER system in which both convergent promoters are activated, whereas lys2 and lacZ transcript levels show little effect on top1Dand top2-1 single mutants, which were dramatically reduced in the double top1Dtop2-1 mutants (20% of wild-type levels after 2 h at 37C). In the pTGlyla system, whereas in wild-type and, to a lesser extent, in the top1Dmutant, the 4-kb transcript levels increased with time at 37C consistent with a faster metabolism at this temperature, they did not change in top2-1 mutants and dropped dramatically in top1Dtop2-1 mutants (Figure 6B). These results were confirmed by analyzing the effect of top2-1 at different temperatures, from 30Cto33 C in which it could be seen a dramatic decrease in the accumulation of lacZ mRNA in the top1Dbackground as the temperature increased (Supplementary Figure S2). Altogether, the results indicate that Topo II plays a critical role in RNAPII transcription in vivo, which indeed is quantitatively more important than that of Topo I. top1Dand top2-1 confer a general hyper-recombination phenotype It is known that top1Dmutants confer a strong hyperrecombination at the rDNA locus (17). To test whether this lack of effect was general for any DNA region and for different types of recombination events and whether it was related to transcription, the effect of top mutations on recombination was analyzed in different and highly sensitive plasmid-borne recombination systems. First we assayed recombination in the pTINV system based on two inverted repeats of the LEU2 gene, one copy of which is a 50-end truncation (leu2D50) and the other a 21-bp insertion mutation under the control of the regulated tet promoter (P tet ::leu2-HOr) (31). This system allows detection of gene conversion events of the leu2-HOr allele with or without crossovers as a function of transcription, which leads to Leu+ recombinants that are scored on SC-leu plates (Figure 7A). As can be seen in Figure 7A, whereas Leu+ recombination levels in top1D and top2-1 mutants were similar to the wild-type both with or without transcription, recombination was increased 26- and 37-fold without or with transcription. Next, we tested the effect on recombination in the pGL-PHO5 system carrying two 0.6-kb leu2 direct repeats under the control of the GAL1 promoter and the PHO5 ORF located between the repeats (32).This system is used to study deletions occurring by recombination between the two leu2 Figure 5. Analysis of the effect of top1Dand top2-1 on DNA supercoiling. (A) Chloroquine gel electrophoresis analysis of plasmid pTGlylaTER in wild-type (HRN1-4A), top1D(TOHR-11A), top2-1 (TORH-6A) and top1Dtop2-1 (TOHR-13C). Strains were cultured under non-transcription conditions in glucose plus doxycycline (Glu +dox) and under transcription conditions in galactose without doxycycline (Gal) at 30C. Electrophoresis was carried out in the presence of 4 mg/ml chloroquine, and hybridization was performed with a labelled LYS2 DNA probe. Southern reveals bands representing topoisomers differing in linking number by steps of one. With the chloroquine concentration used, all topoisomers are negatively supercoiled, with those of increasing negative superhelicity migrating faster in the gel. The band marked as SC consists of the most negatively supercoiled species that are not resolved by the concentration of chloroquine used here. (B) Chloroquine gel electrophoresis analysis of plasmid pTGlyla. Nucleic Acids Research, 2012, Vol. 40, No. 3 1057 Figure 6. Effect of top1 and top2 mutations on transcription of lys-CYCt-lacZ and lys-lacZ fusion constructs at 37C. (A) Northern analyses of lys-CYCt-lacZ-containing mRNAs driven from the tet and GAL1 promoters in wild-type (HRN1-4A), top1D(TOHR-11A), top2-1 (TORH-6A) and top1Dtop2-1 (TOHR-13C) strains at 37C. Mid-log phase plasmid-transformed cells were diluted to an OD 600 of 0.4 synthetic complete (SGal)-Trp medium at 26C and shifted to 37C, samples were taken for northern analyses at different times. (B) Northern analyses of lys-lacZ mRNAs driven from the P tet and P GAL1 promoters. Other details as described in Figure 3. 1058 Nucleic Acids Research, 2012, Vol. 40, No. 3