Role of Transcription in Plasmid Maintenance in the hpr1{Delta} Mutant of Saccharomyces cerevisiae

The Saccharomyces cerevisiae hyperrecombination mutation hpr1 ${Delta}$ results in instability of sequences between direct repeats thatis dependent on transcription of the repeat. Here it is shownthat the HPR1 gene also functions in plasmid stability in thepresence of destabilizing transcription elongation. In the hpr1 ${Delta}$ mutant, plasmid instability results from unchecked transcriptionelongation, which can be suppressed by a strong transcriptionterminator. The plasmid system has been used to examine in vivoaspects of transcription in the absence of Hpr1p. Nuclear run-onstudies suggest that there is an increased RNA polymerase IIdensity in the hpr1 ${Delta}$ mutant strain, but this is not accompaniedby an increase in accumulation of cytoplasmic mRNA. Suppressionof plasmid instability in hpr1 ${Delta}$ can also be achieved by high-copyexpression of the RNA splicing factor SUB2, which has recentlybeen proposed to function in mRNA export, in addition to itsrole in pre-mRNA splicing. High-copy-number SUB2 expressionis accompanied by an increase in message accumulation from theplasmid, suggesting that the mechanism of suppression by Sub2pinvolves the formation of mature mRNA. Models for the role ofHpr1p in mature mRNA formation and the cause of plasmid instabilityin the absence of the Hpr1 protein are discussed.

INTRODUCTION

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Introduction

The hpr1 mutant was originally identified in a genetic screento identify mutants with increased intrachromosomal recombinationevents between direct repeat sequences in yeast (2). The majorityof these events in the hpr1 mutant resulted in a deletion ofthe intervening sequence between the direct repeats, leavingonly one copy of the original repeat (1). Genetic suppressorsof this phenotype cluster in mutations in components of theRNA polymerase II transcriptional machinery. rpb2, sua7 (TFIIB),sin4 (13, 14), srb2 (29), gcr3 (45), and gal11 (31) all suppresshpr1-stimulated hyperrecombination to some degree. This findinghas led to the idea that HPR1 has a functional role in RNA polymeraseII transcription. Indeed, hpr1-stimulated hyperrecombinationhas been demonstrated to be dependent on transcription (13)and specifically dependent on transcriptional elongation (7,32).

Additionally, Hpr1p has been demonstrated to associate withtwo different protein complexes, each further linking HPR1 totranscription. Hpr1p associates with Paf1p, Cdc73p, and Ccr4pas part of an RNAPII complex that is a distinct form of RNApolymerase II, characterized by the absence of Srb proteins(6). Both cdc73 ${Delta}$ and paf1 ${Delta}$ mutants display hyperrecombinationphenotypes, although the phenotypes are not as strong as thatof the hpr1 ${Delta}$ mutant. Consistent with the assembly of Hpr1p intothis RNAPII complex, the hpr1 ${Delta}$ mutation is lethal when combinedwith either a paf1 ${Delta}$ or a ccr4 ${Delta}$ mutation, and the double-mutanthpr1 ${Delta}$ cdc73 ${Delta}$ strain displays temperature-sensitive growth at 30°C.Hpr1p has also been demonstrated to associate with a separateprotein complex, termed the THO complex, that contains Tho2p,Mft1p, and Thp2p (8). The exact function of this complex isnot clear; however, it appears to be involved in linking transcriptionto mRNA export and is associated with the Yra1p, Tex1p, andSub2p factors (41), which have been linked to mRNA export (34,40). Importantly, tho2 ${Delta}$ , mft1 ${Delta}$ , and thp2 ${Delta}$ mutants display the hyperrecombinationphenotype observed in the hpr1 ${Delta}$ mutant and have impaired transcriptionalelongation through the bacterial lacZ sequence (8, 30). It hasbeen proposed that the THO protein complex acts as a functionalunit connecting transcription elongation with mitotic recombination.

Other genetic data suggest that HPR1 in some way influencesthe structural integrity of DNA. First, a full complement oftopoisomerase activities is necessary for viability in hpr1 ${Delta}$ strains (3). Second, the hpr1 ${Delta}$ mutation is lethal when combinedwith a deletion of histones H3 and H4 (hht1-hhf1 ${Delta}$ ) (14). Consistentwith these findings, overexpression of either HHT1-HHF1 or HTA-HTB(H2A-H2B) results in a severe growth defect when combined withan hpr1 ${Delta}$ mutation (51). Overexpression of all four histones hasno effect on the growth of an hpr1 ${Delta}$ mutant strain.

Although there is evidence that Hpr1p promotes transcriptionalelongation, the exact function it performs that allows it topromote elongation is not known and its recent association withmRNA export factors complicates the picture (40, 41). Previousexperiments have demonstrated that Hpr1p-deficient cells havetrouble transcribing through both the bacterial lacZ sequenceand the bacterial ori-amp sequence of pBR322, resulting in theproduction of truncated transcripts (7, 32). Transcriptionalrun-on experiments have also provided some evidence that transcriptionalblocks may be localized to a specific region of lacZ (7). Importantly,when these sequences are located between direct repeats, transcriptionalelongation through them is linked to the recombination eventsobserved between the repeats in hpr1 ${Delta}$ mutants. If elongationcan be stopped prior to entering these sequences with the useof a strong transcription terminator, the recombination phenotypeis not observed (7, 32). Therefore, there appears to be a directlink in hpr1 ${Delta}$ cells between the difficulty in transcribing thesesequences and the hyperrecombination phenotypes observed. However,more recent results suggest that the defective transcriptionof lacZ segments in Hpr1p-deficient cells cannot be correlatedwith a particular DNA sequence or structure (9). Hpr1p seemsto be required for transcription of particularly long DNA sequencesor G+C-rich sequences, and the transcription impairment throughsuch sequences is correlated with their ability to stimulatedirect repeat recombination. This transcription defect can besuppressed by high-copy-number Sub2p (18). Transcription impairmentis associated with a decrease in the steady-state accumulationof full-length mRNA. These observations have been extended todecreased transcription of endogenous DNA sequences that arelong and G+C rich and direct repeat stimulation by these samesequences in an hpr1 ${Delta}$ mutant (9). However, exactly how Hpr1pfunctions in transcription elongation is not understood.

We have reported that hpr1 ${Delta}$ strains have a defect in nuclearmRNA export (34). We have also observed that high-copy expressionof SUB2, a DEAD box family RNA helicase motif-containing geneinvolved in prespliceosome formation (19, 22, 24, 50), suppresseshpr1 ${Delta}$ -stimulated recombination and other hpr1 ${Delta}$ -associated phenotypes(15) and that sub2 mutants have a similar hyperrecombinationphenotype that can be suppressed by high-copy-number HPR1 (15).Interestingly, SUB2 has recently been implicated in nuclearmRNA export of intron-containing and intronless transcripts.

Here we describe a situation in which transcriptional elongationthrough the bacterial ori-amp sequence from a yeast promoterpresent on a CEN-based plasmid contributes to its own instabilityin an hpr1 ${Delta}$ mutant strain. We have used the plasmid loss phenotypeto gain further insight into the transcriptional defects presentin hpr1 ${Delta}$ strains and have examined the effects of high-copy-numberSUB2 on the stability and transcription of the unstable plasmidin hpr1 ${Delta}$ mutant strains to learn more about the mechanism ofsuppression by Sub2p and the role of Hpr1p in transcriptionand mRNA maturation.

MATERIALS AND METHODS

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Materials and Methods

Strains and growth media. The strains used in this study are listed in Table 1. All ofthe strains are isogenic to the W303 RAD5⁺ background. Nonselectivesynthetic complete medium (SC) and SC lacking the indicatedamino acids were prepared by standard methods (37). All strainswere grown at 30°C. For experiments requiring 6-azauracil(6-AU; Sigma), 100 µg of 6-AU per ml was added to liquidSC lacking tryptophan and uracil (SC-Trp-Ura) after autoclaving.

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TABLE 1. Yeast strains used in this study

Plasmid constructions. The plasmids used in this study are described in Table 2. pRM102was constructed by amplifying yeast genomic DNA with primers5'-ATGGATCCGCTGCACGGTCCTGTTCCCTA-3' and 5'-TTGTCGACACCACCAGAACGGCCGTTAGA-3'and inserting the amplified sequence into pRS314 (38) at theBamHI and SalI restriction sites. This sequence includes theentire HIS3 gene, as well as the first 236 bp of the PET56 geneand the first 259 bp of the DED1 gene. The HIS3 promoter inthis amplified sequence was modified such that it containedboth an EcoRI site and a ClaI site inserted between the TATAbox and the translation start site. This modification had nodetectable effect on HIS3 expression and is mentioned solelyto indicate its presence. The pet56 and ded1 sequences are orientedsuch that both promoters directly flank and are directed awayfrom the HIS3 gene. pRM101 was made by inserting the same amplifiedHIS3 sequence into the pGEM-11Zf (Promega) vector with the BamHIand SalI restriction sites. The resulting plasmid was then digestedwith ApaI and SacI. The resulting pet56-HIS3-ded1 fragment waspurified from the digest and then reinserted into pRS314 atthe ApaI and SacI restriction sites, resulting in an inversionof the inserted yeast sequence.

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TABLE 2. Plasmids used in this study

pRM103 is identical to pRM102, except that the ded1 fragment,along with the promoter, has been excised from the plasmid withthe BssHII and SalI restriction sites. It is important to notethat this procedure does not remove the major HIS3 mRNA terminationsite (42). The restriction sites were processed with Klenowfragment to generate blunt ends that were subsequently ligatedtogether. pRM104 is identical to the pRM102 plasmid, exceptthat the HIS3-pet56 region has been removed from the plasmidwith the BssHII and BamHI restriction sites. Again, the overhangingends were processed with Klenow fragment and the plasmid wasreligated at these blunt ends. Therefore, pRM104 contains theded1 fragment together with the DED1 promoter but lacks theHIS3 and pet56 sequences.

pRM110 is pRM102 with a CYC1 transcriptional terminator (26)inserted directly after the ded1 sequence at the SalI and ApaIrestriction sites. The CYC1 terminator was amplified by PCRfrom the yeast genome with primers 5'-CGCGGGTCCTGTCGACCGATATCATGTAATTAGTTATGTCACGCTTAC-3'and 5'-CGCAGGACCCGGGCCCGCGACGATGAGAGTGTAAACTGCGAAGCTTGC-3',which contain SalI and ApaI restriction sites, respectively,at the 5' end. The orientation of the terminator is identicalto that of wild-type CYC1 relative to the direction of transcription.Both of the primers also contain a Psp511 restriction site,which was used to construct pRM111 with the PCR-amplified CYC1transcriptional terminator. pRM111 contains the CYC1 terminatorinserted into the Psp511 restriction site of pRM102, almostdirectly between the Escherichia coli amp and yeast CEN6 sequences.pRM112 has the CYC1 terminator inserted between the ori andamp sequences of pRM102 at the NcoI restriction site.

pRM115 is pRM102 with 630 bp of the E. coli dnaB sequence insertedinto the SalI restriction site. The dnaB sequence was PCR amplifiedwith the primers (each containing a SalI site at the 5' end)5'-CATGGTCGACCCCTTCAACAAACAGCAGGCTGAAC-3' and 5'-CATGGTCGACGTTGAGATCGTCATAACCGGTGTTTAC-3'and inserted into the SalI site of pRM115 such that the sequencewas in the reverse orientation relative to the direction oftranscription. This plasmid was constructed so that the dnaBsequence could be used to probe for plasmid DED1 promoter transcriptionin the presence of high-copy hpr1 suppressor SUB2.

Plasmid loss rates. Plasmid loss rates of strains containing pRM102 (CEN TRP) andits derivatives were calculated as described previously in detail(10). Briefly, cells were grown in liquid SC lacking tryptophan(SC-Trp) to mid-log phase. Equal aliquots were taken and platedonto both SC and SC-Trp plates to determine the percentage ofplasmid-containing cells. The culture was then diluted 1:1,000into liquid SC to release the plasmid selection and allowedto grow to stationary phase. Again, equal aliquots were platedonto SC and SC-Trp to determine the percentage of cells thatstill contained the plasmid. Plasmid loss rates (m) were calculatedwith the formula m = 1 - e^ln(P2/P1)/g, where P₁ and P₂ are thepercentages of plasmid carrying cells before and after the releaseof selection, respectively. g is the number of doublings duringnonselective growth and is described as g = ln(N₂/N₁)/ln2. N₁equals the number of viable cells per milliliter before nonselectivegrowth, and N₂ equals the number of viable cells per milliliterafter nonselective growth (viable cells are cells growing onSC). The significance of differences between the plasmid lossrates was analyzed with the Student t test.

Transcriptional run-on analysis. Transcriptional run-on analysis was performed as previouslydescribed (46), with some modifications (7). Cultures were grownunder plasmid selection in liquid medium to mid-log phase andseparated into two aliquots, one for DNA analysis and one fortranscriptional run-on analysis. For the run-on analysis, 3x 10⁷ cells were chilled by being poured over sterile ice andcollected by centrifugation at 2,860 x g and 4°C. Cellswere then washed in 5 ml of cold (0°C) TMN buffer (10 mMTris [pH 7.4], 100 mM NaCl, 5 mM MgCl₂) and collected by centrifugation.The cells were resuspended in 0.95 ml of 0°C sterile H₂Oand transferred to a microcentrifuge tube. A 0.05-ml volumeof 10% N-lauroyl sarcosine was added, and the tube was lefton ice for 15 min. The tube was gently centrifuged at 4°C(1,830 x g for 1 min), and the supernatant was removed. Thecells were then resuspended in 0.15 ml of a reaction mixturecontaining 20 mM Tris (pH 7.7); 200 mM KCl; 32 mM MgCl₂; 2 mMdithiothreitol; 0.5 mM (each) ATP, CTP, and GTP; and 120 µCiof [³²P]UTP (>3,000 Ci/mmol) and incubated for 7.5 min atroom temperature. The reaction was stopped by addition of 1ml of cold (4°C) TMN buffer containing 1 mM unlabeled UTP.Cells were collected, and RNA was isolated with hot acidic phenol(20).

Probes (5 µg of each) for the run-on analysis were immobilizedto positively charged Hybond-XL nylon membranes (Amersham PharmaciaBiotech) as previously described (11). Each probe was generatedvia PCR amplification and gel purified. The primers used togenerate probes for pRM102 were 5'-CTGTCGTGCCAGCTGCATTAATG-3'and 5'-CCGAAGGGAGAAAGGCGGACAGG-3', 5'-AGCGTGGCGCTTTCTCAATGCTC-3'and 5'-CTCATGACCAAAATCCCTTAACGTGAG-3', 5'-TTATCAAAAAGGATCTTCACCTAG-3'and 5'-ATACACTATTCTCAGAATGACTTGG-3', 5'-GGCGACCGAGTTGCTCTTGC-3'and 5'-GTAAAATCACAGGATTTTCGTG-3', and 5'-TTTACTTATCGTTAATCGAATG-3'and 5'-ATGGTGCACTCTCAGTACAATC-3'.

The rRNA gene control probe (RDN25) used in these experimentswas also generated via PCR amplification with primers 5'-CAACCGGGATTGCCTTAGTAACand TTACGCGTATGGGTTTTACACC-3'. The primers used to generatethe probes for pRM119 were 5'-CAGCTTTTGTTCCCTTTAGTG-3' and 5'-CCGAAGGGAGAAAGGCGGACAGG-3',5'-AGCGTGGCGCTTTCTCAATGCTC-3' and 5'-ATACACTATTCTCAGAATGACTTGG-3',and 5'-GGCGACCGAGTTGCTCTTGC-3' and 5'-ATGGTGCACTCTCAGTACAATC-3'.The ACT1 control probe was generated with the primers 5'-ACTAACATCGATTGCTTCATTC-3'and 5'-TTCTCAAAATGGCGTGAGGTAG-3'.

Isolated RNAs containing [³²P]UTP from the run-on experimentswere hybridized to the filters with the immobilized probes in5x SSC (0.75 M NaCl, 0.075 M Na citrate)-5x Denhardt's solution(0.1% Ficoll 400, 0.1% polyvinylpyrrolidone, 0.1% bovine serumalbumin fraction V)-50% formamide-1% sodium dodecyl sulfate(SDS)-100 ng of single-stranded salmon sperm DNA at 42°Covernight.

Northern and Southern analyses. RNA was isolated from exponential-phase cultures growing inthe appropriate selective medium with hot acidic phenol (20).Northern analysis was performed with formaldehyde agarose gelsby standard procedures (33). RNAs were transferred to and immobilizedon Hybond-XL nylon membranes (Amersham Pharmacia Biotech). Theappropriate probe was hybridized to the transferred RNAs in5x SSC-5x Denhardt's solution-50% formamide-1% SDS-100 µgof single-stranded salmon sperm DNA per ml at 42°C overnight.

Genomic and plasmid DNAs were isolated from yeast in the logarithmicgrowth phase as previously described (37). Southern analysiswas performed in accordance with standard procedures (33). Gelswere transferred to and immobilized on Hybond-XL nylon membranes.The appropriate probes were hybridized to the transferred materialin 5x SSC-5x Denhardt's solution-1% SDS-100 µg of single-strandedsalmon sperm DNA per ml at 68°C overnight.

The DED1 promoter plasmid transcript was detected with a probesynthesized from a fragment (generated by a PCR with primers5'-CTGTCGTGCCAGCTGCATTAATG-3' and 5'-CCGAAGGGAGAAAGGCGGACAGG-3')of the plasmid bacterial ori sequence. This same probe was alsoused to detect plasmid DNA by Southern analysis. To detect theDED1 promoter plasmid transcript with pRM115, a probe was synthesizedfrom the SalI dnaB fragment of pRM115. Detection of actin mRNAon Northern blots, as well as the ACT1 DNA sequences on Southernblots, was done with the same ACT1 sequence as that used forthe run-on analysis. All probes were generated by random primingand labeled with [ ${alpha}$ -³²P]dCTP. All blots, including those fromthe run-on analysis, were visualized with a Bio-Rad MolecularImager FX and quantitated with NIH Image 1.61.

Quantitative RT-PCR methods. Reverse transcription (RT)-PCRs were performed such that thetranscript produced from the DED1 promoter present on pRM102could be detected and quantitated from the five different RTprimers shown in Fig. 5. As a control, each of the RT reactionmixtures contained the same amount of RNA isolated from E. colioverexpressing dnaB. This was done so that the variation betweeneach RT and the subsequent PCR could be measured and properquantitation of the DED1 promoter transcript from pRM102 couldbe achieved. Overexpression of dnaB in E. coli strain BL21(DE3)was achieved with expression plasmid pET3C containing a copyof dnaB (43).

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FIG. 5. Quantitative RT-PCR analysis of transcription from the DED1 promoter of pRM102. (A) Schematic of the locations of the five RT primers used to analyze the DED1 promoter transcript. (B). Southern analysis of the RT-PCR products of the five reactions from wild-type and hpr1 ${Delta}$ cells carrying pRM102. The position of the dnaB transcript used as a PCR efficiency control is indicated. Relative levels of plasmid and total mRNAs present in each reaction were determined by Southern and Northern analyses, respectively. (C). Quantitated RT-PCR levels of hpr1 ${Delta}$ (pRM102) cells relative to those of wild-type cells carrying pRM102 are shown. The results are from three independent experiments for each reaction. The error bars indicate standard deviations. Absolute RT-PCR levels were corrected for the amount of plasmid present in each strain and for the total amount of mRNA. chrom., chromosome.

All yeast cultures were grown to mid-log phase in liquid SC-Trp.Each culture was split in half so that RNA could be isolatedfrom one half and DNA could be isolated from the other. RNAwas isolated with hot acidic phenol (20). DNA was isolated bya standard protocol (37) and used to determine the relativeamount of plasmid present in each strain. RT-PCR was performedwith the Thermoscript RT-PCR System (Life Technologies). TheRT reaction mixture contained 1 µg of total yeast RNA;0.1 µg of total E. coli RNA; 0.2 µM 3' primer; 1xThermoscript cDNA synthesis buffer; 1x Thermoscript RNaseOUT;250 µM each dATP, dCTP, dGTP, and dTTP; and 15 U of Thermoscriptreverse transcriptase. The RNA and 3' primer were first incubatedat 65°C in 10 µl of diethyl pyrocarbonate-treatedwater for 5 min to denature the RNA and the primer. The restof the components were added to create a 20-µl reactionmixture, which was then incubated at 50°C for 60 min. Thereaction was terminated by incubation at 85°C for 5 min.Two units of RNase H was added, and the mixture was incubatedat 37°C for 20 min. Two microliters of this RT reactionmixture was subsequently used for the PCR along with 0.2 µMeach primer; 100 µM each dATP, dCTP, dGTP, and dTTP; 1xmanufacturer's PCR buffer; and 2.5 U of Expand Long Templatepolymerase mixture (Boehringer Mannheim). Normal cycling timeswere 93°C for 30 s, 55°C for 30 s, and 72°C for1 min 30 s. Most reactions were cycled 35 times. Five different3' RT primers were used and designed such that they would hybridizeto the transcript produced from the DED1 promoter present onpRM102. These primers were as follows: RT primer 1, 5'-GTTTCCCGACTGGAAAGCGGGCAGTG-3';primer 2, 5'-CTCATGACCAAAATCCCTTAACGTGAG-3'; primer 3, 5'-ATAGACTGGATGGAGGCGGATAAAG-3';primer 4, 5'-ATACACTATTCTCAGAATGACTTGG-3'; primer 5, 5'-TGTATCCGCTCATGAGACAATAACCC-3'.The dnaB RT control primer, 5'-GCAATTTCCAGCGTACGGTTATCGG-3',was also added to each tube.

The subsequent five PCRs were performed with one each of theabove 3' primers and a single 5' primer (5'-CATATTATGGCTGAACTGAGCGAAC-3')that hybridizes to the DED1 fragment present on pRM102, 110bp downstream from the translation start site. In addition,to each of the five PCRs, specific control dnaB primers 5'-CCCTTCAACAAACAGCAGGCTGAAC-3'and 5'-GCAATTTCCAGCGTACGGTTATCGG-3' were also added.

Exactly the same amount of each PCR mixture was run on 1% agarosegels and subjected to Southern analysis. Each RT-PCR was visualizedwith a radiolabeled probe made from gel-purified PCR amplificationsof the plasmid backbone sequence on pRM102 with primers 5'-GGCCCGGTACCCAGCTTTTG-3'and 5'-GTTTCCCGACTGGAAAGCGGGCAGTG-3' (Fig. 5). dnaB was visualizedwith a radiolabeled probe made from gel-purified PCR amplificationsof dnaB with primers 5'-CCCTTCAACAAACAGCAGGCTGAAC-3' and 5'-GCAATTTCCAGCGTACGGTTATCGG-3'.

Northern analysis probing for actin mRNA was performed on thematerial initially isolated to determine the relative levelsof total RNA. Radiolabeled probe was synthesized from purifiedACT1 PCR amplifications with primers 5'-ACTAACATCGATTGCTTCATTC-3'and 5'-TTCTCAAAATGGCGTGAGGTAG-3'.

The relative amount of plasmid present in each strain was determinedby Southern analysis. Approximately 1.0 µg of DNA fromeach isolation was digested with EcoRI for 4 h at 37°C andresolved on a 1% agarose gel. EcoRI cuts the plasmid once andcuts chromosomal DNA such that a 4.0-kb fragment containingACT1 is produced. DNA blots were simultaneously hybridized withradiolabeled probes designed to recognize plasmid pRM102, aswell as a single-copy chromosomal sequence, ACT1. pRM102 wasvisualized with the same probe used to detect the above-describedRT-PCR products. ACT1 DNA was visualized with the same probeused as described above to detect actin mRNA.

Relative levels of the RT-PCR amplification products were quantitatedand normalized against relative actin mRNA levels, the relativeamount of plasmid present, and the relative efficiency of thednaB RT-PCR. All hybridizations were visualized with a Bio-RadMolecular Imager FX and quantitated with NIH Image 1.61 software.

DNA enzymatic manipulations. All restriction digests were performed in accordance with themanufacturer's recommendations. All PCR amplifications wereperformed with the Expand Long Template PCR System and standardprotocols (Roche Diagnostics). All PCR products were gel purifiedwith 1% agarose gels and the QIAquick Gel Extraction Kit fromQiagen.

RESULTS

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Results

Transcription affects plasmid stability in hpr1. Two plasmids were constructed by inserting a 1.7-kb yeast chromosomalsequence into the multiple cloning site of single-copy yeastplasmid pRS314. The two plasmids differ only in the orientationof the insert, which contains the entire HIS3 gene, along withapproximately 250 bp of the 5' ends of both DED1 and PET56.DED1 and PET56 directly flank HIS3 and are oriented such thatboth promoters are directed away from HIS3 (Fig. 1). These twoplasmids differed markedly in stability when carried by an hpr1 ${Delta}$ strain, while both were stable in the wild-type strain (Fig.1). To quantitate these observations, the rate of loss of eachplasmid in each strain was determined (Table 3). We have previouslyreported plasmid loss rates for pRM102 in both the wild-typeand hpr1 ${Delta}$ strains (15). We repeated these experiments and expandedupon these observations.

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FIG. 1. A yeast chromosomal insert confers differential plasmid stability in an hpr1 ${Delta}$ background, dependent on its orientation in the plasmid. pRM101 and pRM102 differ in the orientation of a 1.7-kb yeast chromosomal insert. The PET56 and DED1 genes are both oriented away from HIS3 and are designated pet56 and ded1 solely to indicate that they are truncations. The photograph above each plasmid diagram gives a qualitative representation of its respective stability in hpr1 ${Delta}$ cells. hpr1 ${Delta}$ cells containing the plasmid were taken from selective (SC-Trp) plates, streaked onto nonselective rich medium (YPD) plates, and then replica plated back onto selective (SC-Trp) plates. An example of a colony that has lost the plasmid is indicated by the white arrow.

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TABLE 3. Plasmid loss rates per cell per generationa

pRM101 has the same stability when it is present in both wild-typeand hpr1 ${Delta}$ cells, and the loss rate in each of these strains isnot significantly different from that of the empty vector (pRS314)alone (Table 3). However, pRM102 is more than eight times decreasedin stability when present in hpr1 ${Delta}$ cells than when present inwild-type cells (P < 0.001). To determine the cause of theinstability of pRM102 in an hpr1 ${Delta}$ mutant strain, we deleted variousregions of the 1.7-kb insert. Deletion of the PET56 fragmentalong with its promoter had no effect on the instability ofpRM102 in an hpr1 ${Delta}$ strain (data not shown). Furthermore, removalof HIS3 with the PET56 fragment from pRM102 (to create pRM104)also did not rescue the instability of this plasmid in an hpr1 ${Delta}$ mutant strain. In fact, pRM104 has an average loss rate of 29.1x 10^-2 and is much more unstable in hpr1 ${Delta}$ cells than is pRM102(P < 0.001) (Table 3). Since pRM104 has only the DED1 fragmentand its promoter inserted in the plasmid, this suggests thatthe main cause of the instability of pRM102 in hpr1 ${Delta}$ mutant cellsis the DED1 promoter. To confirm this, we deleted the DED1 fragment,along with its promoter, from the pRM102 plasmid to create pRM103.This cured the instability of this plasmid in hpr1 ${Delta}$ cells (Table3).

To determine why the DED1 promoter oriented toward the bacterialori-amp sequence in pRM102 causes this plasmid to be unstablein hpr1 ${Delta}$ cells, we examined the transcripts from the DED1 promoterin pRM102 and transcripts from the PET56 promoter in pRM101in both wild-type and hpr1 ${Delta}$ cells (Fig. 2). These Northern assaysindicate that the DED1 promoter is capable of producing an approximately2.5-kb transcript when it is oriented toward the ori-amp sequence(pRM102) in both wild-type and hpr1 ${Delta}$ cells. However, when thePET56 promoter is oriented toward the ori-amp sequence, thislarge transcript is not produced. A 2.5-kb transcript correspondsto a transcript starting from the DED1 promoter and terminatingin front of the CEN6 ARSH4 region of the plasmid. This was confirmedby Northern analysis with probes flanking the CEN6 ARSH4 sequence.Only a probe in front of CEN6 would hybridize to the transcript(data not shown). Therefore, the DED1 promoter present on pRM102is capable of producing a transcript that can progress at leastthrough the bacterial ori-amp sequence on the plasmid. Theseexperiments suggest that the DED1 promoter is relatively strongerthan the PET56 promoter, consistent with previously publishedresults (44).

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FIG. 2. Analysis of transcription through the bacterial ori-amp sequence on both pRM102 and pRM101. Northern analysis was performed to identify transcripts originating from the DED1 promoter (pRM102, lanes 1 through 6) and the PET56 promoter (pRM101, lanes 7 through 12) with a DNA probe from the bacterial ori sequence on the plasmid. RNA was isolated from three independent cultures grown to mid-log phase in selective (SC-Trp) medium for both wild-type (wt) and hpr1 ${Delta}$ strains each containing either pRM102 or pRM101. The arrow points to the major transcript (approximately 2.5 kb) that is generated from the DED1 promoter on pRM102 (lanes 1 to 6). This major transcript is not observed when the PET56 promoter is oriented toward the bacterial ori-amp sequence (pRM101, lanes 7 to 12). Relative actin mRNA levels from each culture are shown at the bottom

The data in Fig. 2 suggest that transcription elongation throughthe ori-amp sequence may be the cause of the plasmid instabilityin hpr1 ${Delta}$ cells. To test this hypothesis, we inserted a strongCYC1 transcriptional terminator at three different sites ofpRM102. First, we inserted the terminator directly 3' to theDED1 fragment on pRM102 to create pRM110 (Fig. 3). Qualitativeobservations indicated that this completely stabilized the plasmidin hpr1 ${Delta}$ cells. Quantitation of the rate of plasmid pRM110 lossconfirmed this and showed that this transcriptional terminatorcompletely suppresses the instability of pRM102 in hpr1 ${Delta}$ cells(Table 3). Northern analysis confirmed that the CYC1 terminatorefficiently blocked all detectable transcription initiatingfrom the DED1 promoter on pRM110 (data not shown). We next insertedthe CYC1 transcriptional terminator directly in front of theCEN6 sequence on pRM102 to create pRM111 (Fig. 3). pRM111 remainedrelatively unstable in hpr1 ${Delta}$ cells compared to pRM110 (Table3) (P < 0.001). Northern analysis of the transcript generatedfrom pRM111 confirmed that transcription progressed throughthe ori-amp sequence and terminated at the CYC1 terminator sequence.Finally, the CYC1 transcription terminator was inserted betweenthe ori and amp sequences to create pRM112 (Fig. 3). Plasmidinstability was suppressed in hpr1 ${Delta}$ cells (Table 3) (P < 0.001).Therefore, termination of transcription from the DED1 promoterprior to progression through the bacterial ori-amp sequenceon pRM102 suppresses the instability in an hpr1 ${Delta}$ strain. However,allowing transcription elongation to pass through the ori-ampsequence and terminate downstream maintains the instabilityof pRM102 in hpr1 ${Delta}$ cells. The finding that the pRM112 plasmidwith a transcription terminator between the ori and amp sequencesis stable in hpr1 ${Delta}$ cells rules out aberrant chromatin changesduring the initiation of transcription as the cause of plasmidinstability.

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FIG. 3. Early termination of transcription from the DED1 promoter suppresses plasmid instability in hpr1 ${Delta}$ cells. CYC1 transcription terminators were inserted either directly after the ded1 sequence on pRM102 (pRM110), directly in front of the CEN6 sequence (pRM111), or between the ori and amp sequences (pRM112) (see Materials and Methods). Given are qualitative assessments of the relative stability of each plasmid in an hpr1 ${Delta}$ background.

Plasmid transcript levels are not reduced in hpr1 ${Delta}$ cells. Previous studies with plasmid-based systems have determinedthat Hpr1p-deficient cells have difficulty with transcriptionelongation through a variety of DNA sequences and often resultin a transcription block that is correlated with genetic instabilitybetween direct repeats (7, 32). If transcription from the DED1promoter on pRM102 in hpr1 ${Delta}$ cells were somehow blocked withinthe ori-amp sequence, then we would expect to see a reductionin steady-state levels of the full-length transcript. The Northernanalysis in Fig. 2 suggests that although the 2.5-kb transcriptcan be detected in hpr1 ${Delta}$ cells carrying pRM102, the levels arereduced compared to those in the wild type. However, properquantitation of this transcript by correction against the amountof plasmid present, as well as the level of actin mRNA and actinDNA, reveals that the levels of the DED1 promoter transcriptare not reduced in hpr1 ${Delta}$ cells compared to those in wild-typecells (Fig. 4). Figure 4 shows the results of three independentexperiments. The absolute levels of the DED1 promoter transcriptare not reduced in hpr1 ${Delta}$ cells relative to those in wild-typecells because the actual levels of pRM102 are very low in hpr1 ${Delta}$ cells, even under selection, because of its inherent instability.On the contrary, these results demonstrate that there mightbe a slight increase in the absolute levels of this transcriptin hpr1 ${Delta}$ cells relative to those in wild-type cells. Althoughthese increases are small, we suspect that they are not trivial.We have observed this slight increase in numerous other Northernexperiments, as well as in quantitative RT-PCR experiments designedto detect levels of the DED1 promoter transcript from pRM102.

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FIG. 4. Quantitation of the major transcript produced from the DED1 promoter on pRM102 in both wild-type (wt) and hpr1 ${Delta}$ strains. (A) Northern analysis of transcription from the DED1 promoter present on pRM102 in both wild-type and hpr1 ${Delta}$ stains. RNA was isolated from three independent cultures of both wild-type and hpr1 ${Delta}$ strains, each containing pRM102, grown to mid-log phase in liquid SC-Trp. The transfers were probed with the same bacterial ori probe as in Fig. 2. The arrow points to the major transcript (approximately 2.5 kb) that is generated from the DED1 promoter on pRM102. (B) Southern analysis to determine the relative amount of plasmid present in each strain. DNA was isolated from each of the same cultures used for the above Northern assay. The DNAs were digested with BamHI, and equal amounts were loaded onto a 0.9% agarose gel. The transferred materials were simultaneously probed for plasmid (with the bacterial ori probe), as well as a single-copy yeast gene (ACT1). (C) Quantitation of the DED1 promoter transcript levels present in hpr1 ${Delta}$ cells relative to those in the wild type. Relative levels of transcript produced from the DED1 promoter were normalized against relative actin mRNA levels, as well as the relative amount of plasmid present in each strain. The relative amount of plasmid was determined by comparing the plasmid levels against that of a single-copy chromosomal sequence (ACT1).

Quantitative RT-PCR was performed to show that the transcriptsobserved in Fig. 4 were derived from the DED1 promoter on pRM102.These experiments also confirmed the quantitation obtained byNorthern analysis. The locations of five RT primers used inthis experiment are shown in Fig. 5. Each primer was used withthe 5' PCR primer to generate five different RT-PCR products,which are shown in Fig. 5B. Quantitation of the results of threeseparate experiments showed that the steady-state level of thepRM102 DED1 promoter transcript in the hpr1 ${Delta}$ strain does notdiffer from that observed in the wild-type strain (Fig. 5C).

Therefore, although we determined that transcription elongationfrom the DED1 promoter on pRM102 is required to observe theplasmid instability in an hpr1 ${Delta}$ strain, we could not detect anyevidence of a transcriptional defect by Northern analysis orRT-PCR.

Plasmid instability in hpr1 ${Delta}$ cells is increased in the presence of 6-AU. To further demonstrate that the instability of pRM102 in hpr1 ${Delta}$ cells is directly related to transcription, we tested the stabilityof the plasmid in the presence of 6-AU. 6-AU decreases intracellularpools of GTP and UTP by inhibiting components of their biosyntheticpathways (12, 17). Consequently, 6-AU sensitivity is often correlatedwith mutations in components of the RNA polymerase II transcriptionalmachinery involved in elongation (17). In the presence of 6-AU,pRM102 was extremely unstable in hpr1 ${Delta}$ cells. The slow growthof this strain made the calculation of a plasmid loss rate impractical.To demonstrate instability, we calculated the fraction of cellspossessing plasmid both in the presence and in the absence of6-AU while the cells were grown in liquid medium selecting forthe plasmid. pRM101 is relatively stable in hpr1 ${Delta}$ cells and isnot affected by the addition of 6-AU (Table 4). Consequently,pRM102 is much more unstable in hpr1 ${Delta}$ cells when 6-AU is presentin the growth medium.

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TABLE 4. Plasmid stability in cultures containing 6AU

High-copy-number SUB2 suppresses plasmid instability in hpr1 ${Delta}$ cells and increases steady-state plasmid DED1 promoter transcript levels relative to those in the wild type. DEAD box-containing putative RNA helicase-encoding gene SUB2,when present on a high-copy-number plasmid, suppresses hpr1 ${Delta}$ -stimulatedrecombination events between direct repeats and the increasedplasmid loss rate of pRM102 (15). This suppression is extremelystrong and results in plasmid loss rates that are close to thewild-type levels. SUB2 suppression is easily visualized withSouthern analysis by comparing the relative plasmid levels inhpr1 ${Delta}$ mutant and wild-type cells (Fig. 6). Since high-copy-numberSUB2 has such a strong stabilizing effect on pRM102 in hpr1 ${Delta}$ cells, we wanted to determine its effect on the steady-statelevels of the plasmid DED1 promoter transcript. For this, weinserted a short unique DNA sequence directly 3' to the ded1sequence on pRM102 (to create pRM115, which is identical topRM102 with regard to plasmid instability in hpr1 ${Delta}$ cells) touse as a probe for the DED1 promoter transcript. We used theunique sequence as a probe since the plasmid containing SUB2(YEp-SUB2) contains the same bacterial ori sequence used toprobe for the pRM102 DED1 promoter transcript. Surprisingly,in the presence of high-copy-number SUB2, hpr1 ${Delta}$ cells displayedsignificantly increased steady-state levels of the DED1 promotertranscript from pRM115 compared to those in wild-type cells(P < 0.01) (Fig. 6). This suggests that the DED1 promoteron pRM115 may actually be more accessible to the RNA polymeraseII transcriptional machinery in hpr1 ${Delta}$ cells. We do not know howhigh-copy-number SUB2 stimulates transcription in an hpr1 ${Delta}$ strain.Presumably, the putative RNA helicase activity is involved inthis process. We have previously shown that Sub2p associateswith RNA polymerase II when it is overexpressed in hpr1 ${Delta}$ cells(15).

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FIG. 6. High-copy-number SUB2 suppresses plasmid instability and increases the relative amount of the major transcript produced from the DED1 promoter on pRM1115 in hpr1 ${Delta}$ cells. pRM115 is identical to pRM102 except that 630 bp of the E. coli dnaB sequence is inserted directly 3' to the ded1 sequence so that the plasmid DED1 promoter transcript could be uniquely probed in the presence of the high-copy-number YEp-SUB2 plasmid (YEp351 backbone). (A) Northern analysis of transcription from the DED1 promoter present on pRM115 in both wild-type (wt) and hpr1 ${Delta}$ stains in the presence of high-copy-number YEp-SUB2. RNA was isolated from three independent cultures grown to mid-log phase in liquid SC-Trp-Leu. The same amount of RNA was loaded onto each lane, and transfers were probed with the bacterial dnaB probe. The blots were subsequently stripped and reprobed for actin mRNA. (B) Southern analysis to determine the relative amount of plasmid pRM115 present in each strain. DNA was isolated from each of the same cultures used for the above Northern assay. DNAs were digested with EcoRI. The transfer was simultaneously probed for pRM115 (dnaB probe) and a single-copy yeast gene (ACT1). (C) Quantitation of the DED1 promoter transcript levels present in hpr1 ${Delta}$ cells relative to those in wild-type cells. Relative levels of transcript produced from the DED1 promoter were normalized against relative actin mRNA levels, as well as the relative amount of plasmid present in each strain.

Transcriptional run-on analysis of plasmid transcripts in hpr1 ${Delta}$ cells. To further elucidate the relationship between transcriptionalelongation through the ori-amp sequence in pRM102 and its instabilityin hpr1 ${Delta}$ cells, we performed a transcriptional run-on analysiswith probes covering the entire ori-amp sequence, as well asthe downstream CEN6-ARSH4 sequence (Fig. 7C). The results ofthese experiments are shown in Fig. 7A. A representative blotis shown on the left, while the averages and standard deviationsof four independent experiments are shown on the right. Theseresults suggest that an increased RNA polymerase II densityis present throughout the length of these sequences in hpr1 ${Delta}$ cells compared to that in wild-type cells. The relative levelson each blot were corrected for both the amount of plasmid presentin the cells and the total amount of RNA (25S rRNA). Transcriptionacross ACT1 was also used to normalize for the total amountof RNA in other run-on experiments with similar results (datanot shown).

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FIG. 7. Transcriptional run-on analysis of the plasmid DED1 promoter transcript in hpr1 ${Delta}$ and wild-type cells. Cells were grown under selection in liquid medium (SC-Trp) to mid-log phase prior to run-on analysis. (A) Radiolabeled RNA from both wild-type (wt) and hpr1 ${Delta}$ cells, each containing pRM102, bound to each probe. Transcription of rRNA was used to normalize transcription levels. The arrow indicates the direction of transcription. The location of each probe from the plasmid is shown in panel C. The bar graph shows the average level of transcription across each probe from four independent experiments for hpr1 ${Delta}$ cells relative to wild-type cells, normalized against both the total amount of RNA (25S rRNA) and the relative amount of plasmid present in each strain (B). Error bars represent the standard deviations. (B) Southern analysis to determine the relative amount of plasmid pRM102 present in each strain. DNA was isolated from each of the same cultures used for the above run-on analysis. DNAs were digested with EcoRI. The transfer was simultaneously probed for pRM102 (bacterial ori probe), as well as a single-copy yeast gene (ACT1). chrom., chromosome.

These results are surprising because previous transcriptionalrun-on experiments have shown a loss of RNA polymerase II densityof transcription of the bacterial lacZ sequences in Hpr1p-deficientcells (7). These same data also provided evidence of a nonspecifictranscription block within this sequence, suggesting that RNApolymerase II transcription may be more susceptible to transientpauses in hpr1 ${Delta}$ . In our experiments, however, we saw an increasein RNA polymerase II density, as well as no evidence of a transcriptionblock when a DED1 promoter was used to drive transcription acrossthe ori-amp sequence.

Plasmid copy number in hpr1 ${Delta}$ cells. One possible explanation for the plasmid instability in hpr1 ${Delta}$ cells is that unchecked transcription might continue throughthe CEN6 region and disrupt its centromere activity. Our Northernanalyses did not show evidence of transcription in this region.However, disruption of the centromere function on pRM102 shouldmake it segregate as a yeast replicating plasmid without a centromere,a YRp plasmid, which is known to be unstable because of missegregation(28, 39). In this model, pRM102 plasmids would accumulate inmother cells, thereby increasing the plasmid copy number incells containing plasmid pRM102. On the basis of our Southernanalyses, we calculated that, under selective conditions, thecopy number of plasmid pRM102 in hpr1 ${Delta}$ cultures is 0.26 ±0.04 of the copy number found in wild-type cultures. From theplasmid loss rate determinations, we found that the proportionof cells in an hpr1 ${Delta}$ culture that carry plasmid pRM102 is 0.28± 0.08. This indicates that there is approximately onecopy of plasmid pRM102 in hpr1 ${Delta}$ cells that have plasmid DNA.This is inconsistent with a missegregation model in which cellsthat contain the plasmid have a greatly increased copy number,up to 50 plasmids per cell.

We also tested a sequestration model of hpr1 ${Delta}$ -mediated plasmidloss. Under this model, a frozen or blocked RNA polymerase II-plasmidDNA-nascent mRNA ternary complex might be complexed with thenuclear membrane such that the sequestered plasmid would notsegregate correctly to the daughter cell. The trapped complexmight make it difficult to recover plasmid DNA by standard isolationmethods, such that the results of Southern analyses done toestimate plasmid copy number are underestimates. To test this,we isolated and quantitated the plasmid copy number by a methodthat can extract DNA from membrane fractions (16). This methoddid not increase the pRM102 plasmid yield in hpr1 ${Delta}$ cells.

Role of homologous recombination in plasmid loss. Another explanation for the plasmid loss in hpr1 ${Delta}$ cells is thataberrant transcription generates a damaged DNA substrate, inthis case, plasmid pRM102, which is a target for homologousrecombination. Recombination between sister chromatids of theplasmid would form a dicentric plasmid, which is known to beunstable because of mitotic nondisjunction events (21, 25).If this were the case, then elimination of recombination througha rad52 ${Delta}$ mutation should increase plasmid stability. We testedplasmid stability in hpr1 ${Delta}$ rad52 ${Delta}$ and hpr1 ${Delta}$ rad50 ${Delta}$ strains and foundthat plasmid stability was unaltered from that observed in anhpr1 ${Delta}$ strain, indicating that increased sister chromatid recombinationis not the explanation for hpr1 ${Delta}$ -mediated plasmid instability.

DISCUSSION

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Discussion

These studies have shown that hpr1 ${Delta}$ cells have defects in thetranscription of certain DNA sequences that can be related toproblems in transcription elongation. These conclusions aresimilar to those of previous studies that found that hpr1 ${Delta}$ cellshave difficulties in transcribing through portions of the bacteriallacZ sequence and parts of the plasmid ori-amp sequence (7,32). The authors of those studies found evidence of transcriptionalstalling, particularly when transcription was driven from thestrong GAL1 promoter under inducing conditions (7). We haveobserved that transcription through the plasmid ori-amp sequenceresults in instability of the plasmid when transcription isdriven by the relatively strong constitutive DED1 promoter.Deletion of the DED1 promoter or insertion of a transcriptionterminator immediately 3' to the promoter restored plasmid stability.

However, in spite of the plasmid instability resulting fromtranscription elongation problems, we did not observe a decreasein message accumulation in hpr1 ${Delta}$ cells by Northern analysis.On the contrary, we observe equal, if not slightly higher, levelsof the destabilizing transcript from the pRM102 DED1 promoterin hpr1 ${Delta}$ cells, while the nuclear run-on experiment with theDED1 promoter showed a relative increase in RNA polymerase IIdensity in hpr1 ${Delta}$ cells. A possible explanation for this observationmay involve our previous finding that polyadenylated RNA exportfrom the nucleus to the cytoplasm is defective in hpr1 ${Delta}$ cells(34, 41). Northern analysis that measures mRNA accumulationwould not reflect the RNA polymerase II transcripts that remaintrapped in the nucleus and are a target for degradation (5,48, 49). The increase in RNA polymerase II density in the hpr1 ${Delta}$ mutant could arise from increased accessibility of the DED1promoter to RNA polymerase II assembly in that promoter region.Increased accessibility in the hpr1 ${Delta}$ mutant could result fromnegative supercoiling at the promoter region behind the multipleRNA polymerase II complexes assembled on a circular DNA templateat a strong promoter. We propose that some of the nascent mRNAtranscripts in the hpr1 ${Delta}$ mutant are incomplete, being eitheraborted or degraded because of aberrant structures. These messageswould not be exported to the cytoplasm. Thus, the increasedtranscription relative to that in the wild type does not resultin increased message accumulation.

We believe that this structural explanation is plausible, giventhe synthetic lethality hpr1 ${Delta}$ displays when combined with eithera top1 ${Delta}$ , a top2(Ts), or a top3 ${Delta}$ mutation (3) or with deletionsin histones H3 and H4 (hht1-hhf1 ${Delta}$ ) (14). Plasmids in yeast canbecome negatively supercoiled in a transcription-dependent mannerwhen they are present in both top1 ${Delta}$ and top1 ${Delta}$ top2(Ts) mutantstrains (4, 35). Indeed, plasmids can become negatively supercoiledin wild-type strains in a transcription-dependent manner giventhe proper conditions of a strong promoter on the plasmid producinga relatively long (7-kb) transcript (27). An elongating RNApolymerase complex generates an accumulation of positive supercoilsin front of the complex and an accumulation of negative supercoilsbehind it (4, 23, 47). It is possible that in an hpr1 ${Delta}$ mutant,localized negative supercoiling may accumulate behind an elongatingRNA polymerase II complex, which in turn would make the correspondingpromoter more accessible.

We have previously reported that high-copy-number expressionof the pre-mRNA splicing component SUB2 suppresses hpr1 ${Delta}$ defects,including instability of plasmid pRM102 (15), and this has beenconfirmed by more recent studies (18). Our studies may givesome insight into the mechanism of suppression. By Northernanalysis, there is a three- to fourfold increase in the pRM102DED1 promoter transcript level in the hpr1 ${Delta}$ mutant carrying ahigh-copy-number SUB2 plasmid, relative to that in the wildtype. This suggests that high-copy-number SUB2 does not suppressby returning transcription to a wild-type level. Rather, high-copy-numberSUB2 appears to make the defective transcripts into productivetranscripts that can be exported to the cytoplasm. The increasein cytoplasmic message accumulation most likely reflects theincreased transcription that occurs in hpr1 ${Delta}$ cells. In a mechanismthat we do not understand, Sub2p overcomes the transcriptiondefect of hpr1 ${Delta}$ cells, allowing production of full-length messagesthat can be processed and transported to the cytoplasm. We wouldexpect nuclear run-on analysis of hpr1 ${Delta}$ plus high-copy-numberSUB2 cells to show the same increase in RNA polymerase II densityas hpr1 ${Delta}$ cells lacking the high-copy-number SUB2 plasmid. However,we have not been able to do this experiment, as the run-on analysisrequires hybridization to the ori-amp sequence and adjacentregions of the plasmid backbone that are also present on theSUB2 plasmid. Nonetheless, we surmise that suppression occursby a resolution of the mRNA processing-export defect of hpr1 ${Delta}$ transcription and not through a bypass of the transcriptionprocess. High-copy-number Sub2p could act as an RNA helicaseto unwind inhibitory secondary structure that may be presentin the nascent RNA molecule. Alternatively, Sub2p could actas an RNA-DNA helicase to unwind the RNA-DNA hybrid just behindRNA polymerase II, thereby allowing the RNA polymerase complexto regress and any blocking structure to be removed. However,in pre-mRNA splicing, Sub2p is not hypothesized to directlycontact the RNA molecule. Rather, Sub2p contacts proteins thatare bound to the 3' splice site region. Sub2p has been proposedto act as an RNPase to aid in the removal of these proteinsfrom the 3' splice site in a protein complex swap (19, 22, 36,50). Sub2p may have a similar role in hpr1 ${Delta}$ suppression by aidingin the removal of a complex from the nascent RNA or the DNAtemplate or the RNA-DNA hybrid region. It will be of interestto see if other hpr1 ${Delta}$ suppressors, in particular the recessivesuppressors that are mutations in the basic transcription apparatus,act in a fashion similar to that of high-copy-number Sub2p bynuclear run-on studies.

We have proposed and tested several models of hpr1 ${Delta}$ -mediatedplasmid instability. The aberrant transcription in an hpr1 ${Delta}$ straindoes not pass through the plasmid centromere region and causeinstability though missegregation. Sister chromatid recombinationto form a dicentric plasmid also seems to be ruled out by ourstudies with recombination-deficient strains. We also foundno evidence of plasmid sequestration in a membrane fraction.The most likely explanation is that the aberrant transcriptionand possible transiently altered plasmid supercoiling in somemanner affects efficient replication at the yeast DNA replicationARS sequence on the plasmid. Once the transcription problemcan be overcome, for example, by high-copy-number SUB2, eventhough the level of transcription remains high, plasmid stabilityis restored. This suggests that it is not the high transcriptionrate per se that affects origin firing but the defective transcription.Perhaps the presence of a trapped mRNA processing-export complex,for example, the TREX complex (41), or trapping of the plasmidin a mRNA export nuclear compartment occludes or prevents theDNA replication complex from assembling on the plasmid. In thisway, a failure of DNA replication would account for the reducedplasmid stability and the low plasmid copy number in cells thathave retained the plasmid.

ACKNOWLEDGMENTS

This work was supported by grant GM30439 from the National Institutesof Health.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biochemistry, NYU School of Medicine, 550 First Ave., New York, NY 10016. Phone: (212) 263-5778. Fax: (212) 263-8166. E-mail: hannah.klein{at}med.nyu.edu.

Present address: Department of Psychiatry, College of Physiciansand Surgeons, Columbia University, New York, NY 10032.

REFERENCES

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