PKD1 Unusual DNA Conformations Are Recognized by Nucleotide Excision Repair*

Albino Bacolla, Adam JaworskiDagger, Timothy D. Connors§, and Robert D. Wells

From the Institute of Biosciences and Technology, Center for Genome Research, Texas A & M University System Health Science Center, Texas Medical Center, Houston, Texas 77030-3303 and § Genzyme Corporation, Framingham, Massachusetts 01701

Received for publication, January 30, 2001, and in revised form, February 23, 2001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The 2.5-kilobase pair poly(purine·pyrimidine) (poly(R·Y)) tract present in intron 21 of the polycystic kidney disease 1 (PKD1) gene has been proposed to contribute to the high mutation frequency of the gene. To evaluate this hypothesis, we investigated the growth rates of 11 Escherichia coli strains, with mutations in the nucleotide excision repair, SOS, and topoisomerase I and/or gyrase genes, harboring plasmids containing the full-length tract, six 5'-truncations of the tract, and a control plasmid (pSPL3). The full-length poly(R·Y) tract induced dramatic losses of cell viability during the first few hours of growth and lengthened the doubling times of the populations in strains with an inducible SOS response. The extent of cell loss was correlated with the length of the poly(R·Y) tract and the levels of negative supercoiling as modulated by the genotype of the strains or drugs that specifically inhibited DNA gyrase or bound to DNA directly, thereby affecting conformations at specific loci. We conclude that the unusual DNA conformations formed by the PKD1 poly(R·Y) tract under the influence of negative supercoiling induced the SOS response pathway, and they were recognized as lesions by the nucleotide excision repair system and were cleaved, causing delays in cell division and loss of the plasmid. These data support a role for this sequence in the mutation of the PKD1 gene by stimulating repair and/or recombination functions.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Polycystic kidney disease (PKD)1 encompasses a family of closely related syndromes characterized by intraparenchymal renal cysts that are lined by a single layer of epithelial cells. The several forms of PKD include autosomal dominant polycystic kidney disease (ADPKD), which is one of the most common inherited human disorders (~1 per 500 worldwide). Affected individuals typically develop large cystic kidneys, but hepatic cysts, intracranial aneurysms, and cardiac valvular abnormalities are extra-renal manifestations often associated with this disorder. Approximately half of ADPKD patients develop end stage renal disease requiring renal replacement therapy and compose ~5% of the chronic dialysis population in the United States (reviewed in Refs. 1-5).

The genetic defect in >85% of ADPKD cases is mutations in PKD1, a gene that encodes a transcript of 14 kilobases from 46 exons spanning 50 kbp on chromosome 16p13.3 (6). The 5' portion of the gene (exons 1-34) is duplicated with more than 95% homology in at least three other copies on chromosome 16p13.1, from which transcripts of 20, 17, and 8.5 kilobases are released (7, 8). However, it is unclear whether these PKD1-like mRNAs are translated into proteins. Polycystin-1, the product of PKD1, is thought to be involved in cell-cell and cell-matrix interactions (9-11) and in calcium-permeable non-selective cation currents (12).

Genotypic analyses of PKD1 microsatellite markers revealed that cysts originate from a single cell that, in a number of cases, underwent loss of heterozygosity. These results led to the proposal that cysts form by a "two-hit" process including a germ line mutation and a subsequent somatic mutation on the functional allele leading to loss of function (13-15). The rate of somatic mutations must be high given the frequent occurrence of ADPKD and the very large number of cysts observed, suggesting the existence of a local hot spot for mutagenesis. The identification of mutations in PKD1 has been hampered by the presence of the homologous genes; several of the 92 mutations identified so far are confined to the 3'-unduplicated region. Therefore, it is unclear whether the frequency of mutations may vary throughout the 50 kbp of the gene (6-8, 13, 17, 89-106).

The PKD1 gene is intersected in intron 21 by an extraordinary 2.5-kbp poly(purine·pyrimidine) (poly(R·Y) tract (16), 1 of the 10 longest sequences of this kind. This poly(R·Y) tract is 66% G·C-rich with 95% C + T in the sense strand and is partly repeated in introns 1 and 22. These poly(R·Y) sequences, which are also present in the PKD1-like homologues, have been proposed to contribute to the high mutation rate of PKD1 (17, 18). A computer search of the 2.5-kbp R·Y sequence revealed 23 mirror repeat sequences, which would be expected to adopt three-stranded DNA structures (intramolecular triplexes) (16) with stems of at least 10 bp. Also, 163 direct repeat sequences were identified, which may adopt slipped, mispaired conformations (16). These structures can block transcription and/or replication and thus induce repair functions (19-23).

Herein, the growth rates of various repair and DNA topological mutant strains of Escherichia coli that harbored plasmids containing the 2.5-kbp poly(R·Y) tract from intron 21 were analyzed. Unusual intramolecular DNA conformations, formed under the influence of negative supercoiling, induced the SOS system and were recognized as lesions by the NER pathway. Delays in cell division were observed as well as loss of the plasmid, suggesting that such DNA conformations were substrates for strand breaks and endonuclease activities.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

E. coli Strains

JTT1 ((gal-25, lambda -, pyrF287, fnr-1, rpsL195(strR), iclR7(Const), trpR72(Am)), RS2:JTT1 (topA10), and SD7:JTT1 (topA10, gyrB226) were obtained from the E. coli Genetic Stock Center at Yale University (New Haven, CT). Strains KMBL1001 (no known mutations), CS5428:KMBL1001 (Delta uvrA::cam), CS5429:KMBL1001 (Delta uvrB::cam), CS5430:KMBL1001 (Delta uvrC::cam), and CS5431:KMBL1001 (Delta uvrD::tet) (24) were obtained from Dr. Nora Goosen (Leiden Institute of Chemistry, The Netherlands). Strains JJC123 (GY6781; lambda  [sfiA::lacZ], Delta pro-lac, gal+, rpsL, mal::Tn9, LexAind1), JJC510 (GY4786; lambda  [p(sfiA::lacZ) CIind-] Delta lac-pro, rpsL), and JJC523 (GY5425; recA441, sulA II, Delta lacI169, thi, leuB6, his4, argE3, ilvTS, galK2, rpsL37, lexA71::Tn5) were kind gifts of Dr. Benedicte Michel (Institut National de la Recherche Agronomique, France).

Plasmids

The cloning of pBS4.0 was reported (16). Plasmids containing truncations at the 5'-end of the 2.5-kbp poly(R·Y) tract were constructed and sequenced by treating pBS4.0 with exonuclease III (Erase-A-base kit, Promega, Madison, WI). The cleavage products were subcloned in pGem-T (Promega), excised as KpnI-SacI fragments, and finally directionally cloned into pBluescript KS- (Stratagene, La Jolla, CA). Six clones were selected for further study, pBS1.8, pBS1.5, pBS1.4, pBS1.3, pBS1.0, and pBS0.8, where the numerals indicate the size of the KpnI-SacI insert (Fig. 2A). The identity of these clones was verified by DNA sequencing (see legend to Fig. 2A). Like pBS4.0, pSPL3 (GenBankTM accession number U19867) is derived from pBluescript and contains, in addition to the vector backbone, coding sequences for the human immunodeficiency virus envelope protein gp160 used for exon trapping (Life Technologies, Inc.).

Chemicals

Novobiocin (Sigma), netropsin (Roche Molecular Biochemicals), and actinomycin D (Sigma) were dissolved in buffer A (0.1 M NaCl, 1 mM sodium phosphate, pH 6.4) at approx 10 mg/ml, and their concentrations were determined spectrophotometrically from their extinction coefficients (epsilon 307 = 6 × 103 (25), epsilon 293 = 2.02 × 104 (26), and epsilon 244 = 2.81 × 104 (25)), respectively.

Growth Curves

E. coli strains were transformed with plasmids by the CaCl2 method (27) and selected on agar plates containing ampicillin (Ap). In all experiments that involved Ap, 100 µg/ml were used. Isolated colonies (one or more depending on size) were transferred to 10 ml of LB medium with Ap and grown to an A600 of approx 0.8-1.1. The number of viable colony-forming units (CFU) from such cultures ("founder cells") was determined by plating dilutions on agar plates without Ap, during which time the 10-ml cultures were kept at 4 °C. This time varied from 1 to 5 days. Control experiments indicated that during this storage time the number of CFU decreased by an average of 33 ± 19%. A known number of CFU was then inoculated into triple-baffled 2-liter fermentation flasks with 1 liter of LB medium pre-warmed to 37 °C, and Ap was added. These flasks were shaken at 250 rpm at 37 °C, and 5-10-ml aliquots were withdrawn after 5 min (t0) and subsequently at every 15-30 min for 8-10 h. To determine the number of CFU/ml, the aliquots were diluted in LB and plated on agar plates without Ap.

The following parameters were determined for each growth curve.

The Doubling Time-- The doubling time (t2) during the exponential phase of growth was obtained from the relation t2 = ln 2/k, where k was the slope as shown in Equation 1,


<UP>ln CFU/ml</UP>=<UP>ln</UP>(<UP>CFU/ml</UP>)<SUB><UP>0</UP></SUB>+kt (Eq. 1)
The standard error of t2 (Et2) was derived from the standard error of k (Ek) according to Et2 t2Ek/k.

The Maximum Fold Decrease in CFU/ml (MDC)-- During the first few hours of growth, the number of CFU/ml either decreased or increased non-exponentially. These data were fit by high degree polynomials. MDC was obtained by dividing the CFU/ml at time 0 ((CFU/ml)t0) by the CFU/ml at the curve minimum ((CFU/ml)t(min)). When there was no decrease in CFU/ml, (CFU/ml)t(min) = (CFU/ml)t0 and MDC = 1.

Duration of the Non-exponential Phase of Growth (tA)-- We define tA as the time it would have taken for the bacterial population to reach (CFU/ml)t0 if it grew exponentially from the theoretical (CFU/ml)0 given by Equation 1. Accordingly, tA = ln((CFU/ml)t0/(CFU/ml)0)/k.

Topology of Closed Circular DNA

The following relations were used to analyze the superhelical density of plasmids. For relaxed, circular DNA Lk0 = Tw0 + Wr0, where Tw0 = n/h0, n is the total number of bp and h0 the helical pitch.

Addition of a ligand that binds DNA and alters h0 (twist) gives Lk0t + Wr0t under conditions where DNA is relaxed (i.e. by topoisomerase I). Subsequent ligand removal in the absence of strand breakage yields Lk0t = Tw0 + Wrt, where Wrt = Wr0 + tau t, and tau t is the number of ligand-induced superhelical turns. For an in vivo population of negatively supercoiled topological isomers, <Lk> = Tw0 + <Wr> (<Wr> = Wr0 + <tau >, where tau  is the number of negative supercoils). Addition of a ligand in vivo in the presence of homeostatic control of negative supercoiling, followed by its removal in the absence of strand breaks, yields <Lk> = Tw0 + <Wrt>. This population of topoisomers will have, on average, fewer negative supercoils when the ligand increases twist and more negative supercoils when the ligand decreases twist (28, 29).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Sequence Analysis of the Poly(R·Y) Sequence-- A previous computer search performed on the 2.5-kbp poly(R·Y) identified the presence of 23 perfect mirror repeats, which can form DNA triple helices (16). We find that these motifs are clustered into three distinct regions separated by ~600 and 450 bp, respectively (Fig. 1). A search for perfect tandem repeats (30), which will form slipped structures (31, 32), revealed more than a thousand. The dinucleotides, TC and CT (row a), are most common; however, they are excluded from the 5'-end where the mirror repeats predominate. These dinucleotides are found with increasing frequency toward the 3'-end. The trinucleotide repeats are mostly CCT and are localized within the 5'-half of the tract, with the exclusion of the very 5'-end (row b). Pentanucleotide repeats are of three types as follows: CTCCC, CTCCT, and CCCAT. The first type (row c) can be further subdivided into three clusters according to the following reading frames: 8 CTCCC tandem repeat units (TRU) at the 5'-end, 5 CCTCC TRU in the middle, and a cluster of 19 TCCCC TRU at the 3'-end. The CTCCT direct repeats are positioned exclusively in the middle of the tract, interspersed by a single CCCAT tandem repeat (row d). Longer direct repeats are common as shown in row e. The tract also contains close runs of guanines throughout its length that may form four-stranded structures (G-quartets) (33).


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Fig. 1.   Sequence motifs of the 2.5-kbp poly(R·Y) tract. The boxes labeled Mirror Repeats (16), Tandem Repeats, and P1 Nuclease sites (19) indicate the length of the 2.5-kbp poly(R·Y) tract from intron 21 of the PKD1 gene. Row a identifies dinucleotide direct repeats (CT and TC); row b, CCT direct repeats; row c, CTCCC, CCTCC, and TCCCC direct repeats; row d, CTCCT direct repeats in red, and one CCCAT direct repeat in black; row e, ATCCCTCTCCC direct repeats in black, TCCCCCTTCTC in red, CCTCTTCCCCTCCCCT in yellow, CTCCTCTCCTCCCCA in light blue, TTCCCCTCCCCTCCTC in green, and CCCTCCCCTCTCCCCCTTCTCTC in purple.

In summary, although many mirror and direct repeats are present, their locations are clustered, suggesting highly specific evolutionary duplication events. To the best of our knowledge, this tract contains the highest density of unorthodox simple sequence repeat features (mirror, direct repeats, and R·Y strand bias) of any known sequence of this length.

Previous analyses revealed that under superhelical stress the 2.5-kbp poly(R·Y) tract was cleaved by the single-stranded specific nuclease P1 at four locations, suggesting the formation of unusual structures (19). The location of the four P1 nuclease-sensitive sites is not coincident with the position of the direct repeats with <= 5 bp (Fig. 1); however, sites 1 and 2 do superimpose with clusters of overlapping mirror repeats, as well as with two of the longest direct repeats. This suggests that the unusual DNA structures formed by the poly(R·Y) sequence in vitro include intramolecular triplexes and mispaired loops.

Length-dependent Delay in Cell Growth-- To determine whether such structures form and influence cell physiology, we investigated the effect of the full-length tract and its truncations (Fig. 2A) on the growth rate of wild-type E. coli KMBL1001. Fig. 2B shows the normalized growth curves obtained with each of the plasmids. The doubling time was lengthened in proportion with the length of the inserts from 21.9 min for pBS0.8 to 24.4 min for pBS4.0. Four plasmids (pBS4.0, pBS1.8, pBS1.5, and pBS0.8) caused a decrease in CFU/ml during the first few hours of growth (Fig. 2B, inset). This loss of viable cells was largest for pBS4.0 that had the full-length poly(R·Y) tract, and it decreased in proportion to the length of the inserts. Finally, the duration of the non-exponential phase of growth (lag period) also lengthened in accordance with the increasing insert lengths, from 0.8 h for pBS0.8 to 1.8 h for pBS4.0.


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Fig. 2.   Truncations of the poly(R·Y) sequence. A, list of plasmids containing truncations in the poly(R·Y) tract. Solid lines, genomic sequences; open rectangles, the original 2.5-kbp poly(R·Y) (pBS4.0) or exonuclease III deletions from the 5'-end (pBS1.8 through pBS0.8) ("Experimental Procedures") (16). The 5'-end of pBS4.0 coincides with the BamHI site located in intron 18 of the PKD1 gene, whereas the 3'-end of all clones is a SacI site located in exon 22. The 5'-end of pBS1.5, pBS1.4, pBS1.3, pBS1.0, and pBS0.8 is a KpnI site. The lengths of the inserts in bp are as follows: pBS4.0, 4064; pBS1.8, 1765; pBS1.5, 1493; pBS1.4, 1357; pBS1.3, 1270; pBS1.0, 1008; and pBS0.8, 850 (see "Experimental Procedures"). B, growth curves of E. coli KMBL1001 harboring pBS4.0 through pBS0.8. Growth curves were normalized by dividing the CFU/ml at time = 0 ((CFU/ml)t0) and at subsequent times ((CFU/mlt)) by (CFU/ml)t0. The numbers of CFU/ml used to start the 1-liter cultures are as follows: pBS4.0, 3200 (filled circles); pBS1.8, 3700 (open circles); pBS1.5, 3100 (squares); pBS1.4, 2800 (triangles); pBS1.3, 1600 (inverted triangles); pBS1.0, 2500 (diamonds); and pBS0.8, 2400 (hexagons). Inset, expanded version of the data at the early time points. Linear regressions of the data (in the order reported above) for MDC, t2, and tA gave r2 values of 0.68, 0.46, and 0.87, respectively.

Thus, we conclude that the poly(R·Y) sequence compromised the viability of a number of cells in a length-dependent manner and contributed to the lengthening of the population doubling time as well as the lag period, suggesting that the magnitude of these effects correlated with the number of unusual DNA structures that formed.

DNA Topology, Repair Functions, and SOS Response-- To identify genetic factors associated with cell death, growth curves were determined for E. coli mutant strains that affected DNA supercoil density, nucleotide excision repair (NER), and DNA damage-induced SOS response (Table I). The parameters (MDC, t2, and tA) obtained in E. coli strains harboring pBS4.0 were compared with those obtained for pSPL3, a control chosen because of its size (6.0 versus 7.0 kbp for pBS4.0), the absence of long repetitive sequences, and because, like pBS4.0, it was a derivative of pBluescript.

                              
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Table I
E. coli strains

The results (Table II) reveal the following. First, the extent of cell loss mediated by pBS4.0 correlated with the steady-state level of negative supercoiling (as determined in vivo for pUC19, not shown) because the magnitude of MDC followed the order topA10 wt > topA10gyrB226. Since the number and stability of underwound unusual DNA structures increase with the extent of negative supercoiling (20-23, 28, 34, 35), we conclude that supercoiling stabilized their formation and that this was critical for the loss of cell viability. Furthermore, the differences in doubling time between pSPL3 and pBS4.0 in the three top/gyr strains (15.3, 9.3, and 5.4 min, respectively) also depended upon negative supercoiling, suggesting that the formation of unusual DNA structures interfered with cell division.

                              
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Table II
Parameters for the growth curves
MDC = maximum fold decrease in CFU/ml (MDC = 1, no decrease in CFU/ml); t2 = doubling time during exponential growth; tA = lag period (time required to reach the equivalent of initial CFU/ml for exponential growth extrapolated to time = 0). The standard error for t2 ranged from 0.3 to 3.7 min. The variations in MDC and tA are discussed under "Results."

Second, E. coli NER mutants revealed that UvrB and UvrC were necessary for the loss of cells. In fact, the MDC was negligible for both Delta uvrB and Delta uvrC strains compared with their isogenic wild-type KMBL1001. In addition, for Delta uvrB there was no lengthening of the doubling time, whereas Delta tA was shortened to ~15 min from >= 1.3 h observed for all the other strains. The second significant result from this analysis was the considerable loss of cells in the Delta uvrA and Delta uvrD strains harboring only the control plasmid pSPL3. Because deficiency in UvrD is known to lead to constitutive expression of the SOS response (36-38), a likely explanation is that the SOS system may induce apoptosis during the first period of cell population growth.

Third, we investigated the E. coli SOS strains. During the preparation of JJC523 (Delta lexA71) "founder cells" harboring pBS4.0, we observed significant cell lysis that yielded ~1 × 105 CFU/ml from overnight cultures compared with >1 × 107 obtained normally with the same strain harboring pSPL3 or all the other strains with either pBS4.0 or pSPL3. This result clearly indicates that pBS4.0 caused extensive cell death when associated with a fully active SOS response. The growth curve started from the surviving cells did not show a large decrease in cell count. The MDC value with pBS4.0 for the wild-type JJC510 was much lower than for the other two wild-type strains, JTT1 and KMBL1001. We observed that the values of MDC (and therefore tA) for the three wild-type strains, for topA10, and for topA10gyrB226, varied considerably between experiments. Further analyses indicated that most of this variability arose during the preparation of founder cells used to start the growth curves. At that stage a variable number of cells lost their plasmid; these cells then succumbed at the beginning of the growth culture, when MDC was measured. In the lexAind1 strain JJC123 harboring pBS4.0, where the SOS system is not inducible, the MDC value was comparable to that of its isogenic wild-type strain, JJC510.

In summary, the data indicate that the poly(R·Y) sequence from intron 21 of PKD1 formed unusual DNA structures as a consequence of negative superhelical density and that the detrimental effects on growth depended upon activation of the SOS system. Furthermore, both UvrB and UvrC were necessary to elicit the pBS4.0-dependent lethality.

Integrity of the poly(R·Y) Sequence in Surviving Cells-- Previous work showed that long repetitive sequences such as (CTG·CAG)n and (CGG·CCG)n are unstable and that the average length of the repeats shortens as the cell population ages (39-42). Therefore, we investigated whether the surviving cells represented a subpopulation that had deleted part, or all, of the poly(R·Y) tract. pBS4.0 was isolated from all of the strains and analyzed in two ways. First, the 4.0-kbp PKD insert was excised from the vector, and the insert and vector were separated by agarose gel electrophoresis. Most lanes showed considerable smearing throughout their length. However, no discrete products were visible besides the two expected bands, except 1 sample out of 33 where a recombination event took place. Second, these inserts and vectors were radioactively labeled, and their molar ratio was calculated. The ratios ranged from 0.47 ± 0.07 to 0.64 ± 0.06; however, no statistical significance was found among the mean values, and no correlation was observed between these ratios and the MDC values obtained for the same E. coli strains.

Thus, we conclude the following. First, loss of the full-length poly(R·Y) tract in favor of more stably transmitted deletion products was not observed. Second, both the smearing and the lower molar ratio of the PKD insert indicate that a proportion of pBS4.0 was in the process of being degraded at the time of plasmid isolation and that the starting point for such degradation was within the PKD insert. We suggest that pBS4.0 was either replicated and transmitted intact during cell division or cleaved at the poly(R·Y) tract and then rapidly degraded.

Influence of Negative Supercoiling-- To verify further the differences observed in the topoisomerase I and gyrase E. coli mutants, as well as the Delta uvrB strain, MDC was evaluated in identical cells following alterations in their levels of negative supercoiling. We used novobiocin to inhibit the assembly of active gyrase (43, 44) and thus achieve a relaxation of the DNA in vivo. This should reduce the formation of unusual DNA structures and relieve the extent of founder cell loss. Fig. 3 shows that the population of more highly negative supercoiled topoisomers of pUC19 was progressively reduced in the presence of novobiocin, as expected.


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Fig. 3.   Negative superhelical density in vivo. Topoisomers of pUC19 were isolated from E. coli strain KMBL1001 grown to A600 of 0.5 in the absence or in the presence of 5 µM novobiocin and analyzed by agarose gel electrophoresis (1% w/v) in 90 mM Tris borate, 2 mM EDTA, pH 8.0, in the presence of chloroquine. The gel negatives were scanned with a PhosphorImager and the data were smoothed by a low pass filter that used a fast Fourier transform to remove abnormal high frequencies. The y axis is the area of each peak in pixels as given by the PhosphorImager. The negative superhelical density (x axis) was obtained as described (35). Data were fit to a three-parameter Gaussian curve. Filled circles, no drug; open circles, with drug.

Fig. 4A shows the growth curves for the topA10 strain carrying pBS4.0 in the absence or presence of novobiocin. Addition of novobiocin during the preparation of cells maintained their complete viability in the subsequent growth, whereas >90% was lost in its absence. Superimposable growth curves were found for 2-10 µM novobiocin. Cell loss was not prevented when novobiocin was added at the beginning of the growth curves rather than during the preparation of founder cells, indicating that the loss required an activity to take place at an early step.


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Fig. 4.   Growth curves for E. coli strains transformed with pBS4.0 prepared in the presence of novobiocin, actinomycin D, or netropsin. E. coli cells were transformed with pBS4.0 and selected on Ap plates. A few colonies were suspended in 1 ml of LB medium, and 100-200 µl was transferred to several tubes containing 10 ml of LB medium with Ap and various concentrations of novobiocin or actinomycin D or netropsin. The cultures were grown overnight, and the number of CFU/ml was determined by plating aliquots on agar plates without Ap. A known number of CFU/ml was then transferred the next day to 1-liter cultures with Ap but without novobiocin or actinomycin D or netropsin, and the growth curves were determined. A, E. coli topA10 and 5 µM novobiocin; B, E. coli KMBL1001 and 30 µM actinomycin D; C, E. coli KMBL1001 and 4 µM netropsin; D, E. coli Delta uvrB and 4 µM netropsin. Filled circles, no drug; open circles, with drug. Only the data points from the early times are shown. Note that the x and y axes are not identical in the four panels. The Delta t2 values (t2drug - t2control) for A-D were 3.2, -0.9, 0.5, and -0.2 min, whereas the ratios MDCdrug/MDCcontrol were 0.05, 0.05, 16.7, and 0.8.

An alternate strategy was also implemented to influence supercoiling, and hence DNA structure, by preparing cells in the presence of drugs (actinomycin D or netropsin) that bind directly to DNA and thereby influence its global topology as well as its conformations at specific sequences. Analyses of the topoisomer distributions of pUC19 in the presence of either drug confirmed their binding to the DNA (not shown).

Actinomycin D intercalates at G·C pairs, and it reduces the number of negative superhelical turns and stabilizes DNA in the right-handed duplex B-form (45-50). Due to its G-C richness, the poly(R·Y) tract offers numerous binding sites for the drug, whose activity is expected to decrease the number of inviable cells.

Fig. 4B shows the growth curves for the wild-type E. coli strain KMBL1001 harboring pBS4.0 in the absence or presence of actinomycin D. As described for novobiocin, actinomycin D was only added during the preparation of cells. The extent of cell loss within the first 2 h decreased progressively in the presence of 5-30 µM actinomycin D, supporting the hypothesis that the formation of underwound non-B DNA structures was responsible for cell loss.

Netropsin binds to the minor groove of A·T pairs, requiring four or more such bp for optimal contacts, introduces additional negative supercoils, destabilizes triplex DNA, and increases the stiffness of the double helix (26, 51-58). We reasoned that netropsin would accentuate cell loss in two ways. First, it would bind to duplex B-DNA and induce additional negative supercoiling. Second, it would bind selectively to the vector sequences (of the 131 consecutive four A-T pairs only 7% are in the poly(R·Y) tract) and thus increase the stiffness of the vector. Thus, the superhelical density would be preferentially partitioned into the poly(R·Y) tract, which is the most flexible and writhed region of the plasmid (59).

Fig. 4C shows the growth curves for wild-type E. coli KMBL1001 carrying pBS4.0 in the absence and presence of netropsin. As described above, the drug was only added during the preparation of cells. The data show that netropsin strongly accentuated cell selection during the first 5 h. The growth curves were not affected by the addition of netropsin to E. coli KMBL1001 cells harboring pSPL3 or when the compound was added to E. coli KMBL1001 containing pBS4.0 only at the beginning of the growth curve culture.

To verify further whether cell loss was caused by the formation of non-B DNA structures alone or whether it required their recognition by the NER system, the experiment with netropsin was conducted in E. coli Delta uvrB carrying pBS4.0. Contrary to the wild-type strain, Delta uvrB cells were unaffected by the drug (Fig. 4D), proving that the recognition of unusual DNA structures by NER was indispensable for eliciting cell selection.

In summary, we conclude that optimal viability required the DNA to be maintained in an orthodox right-handed B-form. We also conclude that the reactions of the NER proteins on the underwound non-B DNA structures, but not their formation alone, was indispensable for eliciting loss of cell viability.

Loss of Plasmid-- The relationship between the SOS and NER pathways and cell viability became apparent when colonies derived from the exponential phase of the growth curves were analyzed. Samples of E. coli transformed with pSPL3 taken throughout the period of growth yielded colonies of essentially homogeneous size when plated without Ap. Alternatively, colonies derived from several E. coli strains transformed with pBS4.0 plated after the cultures grew for 3-5 h appeared heterogeneous in size. DNA isolated from small and large colonies revealed that pBS4.0 was only present in the small but not in the large colonies.

To determine the extent of plasmid loss for pSPL3 and pBS4.0, the number of Ap-resistant (ApR) and Ap-sensitive colonies for various E. coli strains was measured. Fig. 5A shows the logarithm of the ratio between the number of ApR colonies and the total number of colonies for E. coli strains harboring either of the two plasmids. For pSPL3 (Fig. 5A, hatched bars), the greatest differences were observed among the wild-type strains, whereas only small differences were seen between the wild-types and their respective mutants. Ten and 90% of the JTT1 and JJC510 cells, respectively, contained the plasmid, whereas less than 0.1% of cells retained pSPL3 for the KMBL1001 strain. Retention of pSPL3 was slightly greater (2-5-fold) in the Delta uvrB and Delta uvrC mutants (the Delta uvrA and Delta uvrD mutants were not tested). Addition of netropsin to KMBL1001 cells caused the dramatic reduction by 4 orders of magnitude in the number of plasmid-containing cells, whereas addition of actinomycin D or novobiocin slightly increased the retention of pSPL3 5- and 10-fold, respectively.


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Fig. 5.   Loss of plasmid DNA and viability of untransformed cells. A, the strains designated were transformed with the pSPL3 vector or with pBS4.0. Cells were prepared as for the growth curves and used to start overnight cultures in LB medium containing Ap. The y axis is the logarithm of the ratio between the number of colonies formed by plating dilutions of the overnight cultures on plates containing Ap (ApR) and the number found on plates without Ap (ApR+S). A value of 0 indicates no loss of plasmid. E. coli strains are from left to right as follows: KMBL1001, Delta uvrB, Delta uvrC, KMBL1001 grown in the presence of 4 µM netropsin (net), KMBL1001 grown in the presence of 30 µM actinomycin D (act), KMBL1001 grown in the presence of 5 µM novobiocin (nov), JJT1, topA10, topA10gyrB226, JJC510, lexAind1, Delta lexA71. Filled bars, pSPL3; hatched bars, pBS4.0. wt, wild type. B, untransformed cells were grown overnight in LB medium and then stored for 4 days at 4 °C with the addition of Ap. Very limited loss of viability was observed during this period. A fresh 1-liter culture (with Ap) was then started and the number of colonies counted on agar plates without Ap for the first 4 h. Filled circles, E. coli JTT1; filled triangles, E. coli JJC510; open circles, E. coli KMBL1001; open triangles, E. coli Delta uvrB.

These results indicate that plasmid stability was improved when negative supercoiling was lessened, as expected. The topA10 mutation increased pSPL3 instability by about 5-fold, whereas the stability increased 8-fold in the topA10gyrB226 strain, confirming that supercoil tension was detrimental to plasmid stability. Inactivation or constitutive expression of the SOS system in the Delta lexA71 and lexAind1 strains, respectively, did not affect the retention of pSPL3.

In summary, these results with pSPL3 show that plasmid loss commonly took place during cell growth, that the extent of loss depended on the host genetic background, and that it correlated with negative supercoil density.

As for pSPL3, pBS4.0 was least stable in wild-type KMBL1001 as compared with JTT1 and the JJC510 strains. However, its retention was reduced relative to pSPL3 by 1000-, 10-, and 1000-fold in KMBL1001, JTT1, and JJC510, respectively, indicating that the PKD1 insert was detrimental to plasmid stability. Significantly, retention of pBS4.0 improved by nearly 6 orders of magnitude in the Delta uvrB mutant strain, strongly suggesting that loss of pBS4.0 was mediated by NER. Deletion of the uvrC gene or addition of netropsin to KMBL1001 did not further increase the number of cells without pBS4.0, whereas growth of KMBL1001 cells both in the presence of actinomycin D or novobiocin increased the retention of pBS4.0 by 4 and 2 orders of magnitude, respectively. The topA10 mutation decreased the number of cells containing pBS4.0 by 10,000-fold, whereas the topA10gyrB226 mutation increased their fraction 10-fold. Because both the genetic and pharmacological approaches agreed that loss of pBS4.0 was greatly dependent on negative supercoiling, we conclude that alternative DNA structures in the poly(R·Y) tract were critical for leading to plasmid loss through their interaction with the NER pathway. Inactivation of the SOS system in the lexAind1 strain increased pBS4.0 loss about 10-fold. The results with the Delta lexA71 cells were difficult to interpret because of the substantial lysis observed in the presence of pBS4.0, as noted above.

Hence, this analysis suggests that (i) plasmid loss is a general phenomenon that depends on the host genetic background; (ii) negative supercoiling increases the propensity of losing a plasmid; (iii) the PKD1 insert in pBS4.0 greatly increases the propensity to lose the plasmid; (iv) the UvrB component of the NER system is required for the PKD1 sequence-mediated plasmid loss; and (v) constitutive induction of the SOS system leads to cell lysis in the presence of pBS4.0.

To determine whether cell loss during the growth curves (Table II) could be explained by the behavior of cells that lack plasmid, untransformed wild-type E. coli JTT1, JJC510, KMBL1001, and the Delta uvrB mutant were prepared in the absence of Ap and then used to mock-start a growth curve in fresh medium with Ap. Fig. 5B shows that >98% of these cells lost their viability within the first 2 h of growth. This result is consistent with the hypothesis that cell death was mediated by the selection stress on cells that had lost their plasmid during the previous culture.

In summary, these data show that the poly(R·Y) sequence from intron 21 of the PKD1 gene contains unusual DNA structures that form under the influence of negative supercoiling, that these structures interact with components of the NER pathway, and that such interactions lead to plasmid loss.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Prior studies with simple repeating sequences ((GC·GC)n or (TG·CA)n) that in vitro adopt the left-handed Z-DNA conformation (60-63), oligo(R·Y) tracts (e.g. (GAA·TTC)n), which form intramolecular triplexes (64-66), or triplet repeat sequences (i.e. (CTG·CAG)n and (CGG·CCG)n) that are particularly flexible and form slipped structures (see Ref. 59 and reviewed in Ref. 67) revealed considerable instabilities. Also, the in vivo existence of left-handed Z-DNA (61, 68) and triplexes was documented (63, 69, 70). It was hypothesized (60-67), but not proven, that these instabilities were due to the capacity of these sequences in vivo to adopt non-B DNA conformations. Herein, we provide genetic evidence that the unusual conformations and not the sequences per se are responsible. Several cellular factors (i.e. NER, the SOS system, and gyrase and topoisomerase I functions) are involved, which interact with or influence the stability of the unorthodox DNA structures.

These data are the first to link directly DNA conformations and their polymorphic behaviors with mutagenesis in vivo. The capacity of certain types of DNA sequences to undergo facile transformations from right-handed B structures to unorthodox conformations (i.e. triplexes, slipped structures, etc.) is an integral component of these studies.

The role of DNA topology (supercoiling) was evaluated with mutant strains and with a gyrase inhibitor (novobiocin) that affected supercoil density as well as with ligands (actinomycin D and netropsin) that bind directly to DNA and thereby influence its global topology and hence its conformations at specific sequences. The composite data show that unusual DNA structures (triplexes and slipped structures) in the 2.5-kbp poly(R·Y) tract from the PKD1 gene exist in E. coli and are responsible for the observed genotoxicity. The PKD1 gene is a human sequence on chromosome 16p13.3, and hence the types of structural transitions formed in this plasmid system may also occur in humans.

Fig. 6 outlines the main steps that may be involved in the process. The in vivo homeostatic control of negative supercoiling (71-73) (Fig. 6, A and B) generates plasmid topoisomers in which plectonemic conformations maintain the DNA under highly negative torsional stress (74, 75). The poly(R·Y) tract undergoes structural transitions (Fig. 6C) to various non-B structures (19-23) under the influence of both steady-state levels of negative supercoiling and waves of hyper-negative supercoiling generated by the passage of DNA helix-tracking enzymes such as during transcription, replication, or repair. Underwound unusual DNA structures should be dissipated as they are approached by an incoming polymerase due to positive supercoiling formed ahead of the complex (76-79) and thus are predicted to not interfere with replication. E. coli strains defective in the SOS-induced genes, specifically Delta uvrA and Delta uvrB (80), failed to show a lengthening of the population doubling time, in contrast with the strains with a functional SOS system. Thus, unusual DNA structures may have interfered with cell division only after DNA damage (i.e. strand-break(s)) inflicted on them by NER (81) caused replication forks to collapse (82-85) (Fig. 6, D and E).


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Fig. 6.   Model for supercoiling mediated selection against cells that carry the poly(R·Y) sequence. Open arc, the 2.5-kbp poly(R·Y) sequence; gray symbols, mirror and direct repeats capable of forming unusual DNA structures; thin line, vector DNA; open squares and triangles, mirror and direct repeats in duplex B structures; filled symbols, non-B DNA structures sustained by negative supercoiling; hatched ovals, damage recognition and incision complex of the NER system.

Experiments performed previously with plasmids containing (CTG·CAG) triplet repeats (39, 67, 86, 87) showed that the tract frequently deletes to discrete and stably transmitted new species in E. coli populations and that both the MMR and NER systems are involved (67, 86, 87). On the contrary, we show here that the poly(R·Y) tract from the PKD1 gene does not give rise to such discrete and stably transmitted deletions. A possible reason for the difference is that the presence of the triplet repetitive elements within the (CTG·CAG) tracts facilitates post-DNA damage repair and/or recombination and thus maintains plasmid viability, which is lost when similar damage is inflicted within the poly(R·Y) tract. We also show (Fig. 4) that the NER nuclease activity related to DNA stability is not directed to a particular DNA sequence but rather to DNA structures that may arise from several types of sequences. Therefore, we conclude that the NER system has the capacity of recognizing certain non-B DNA structures formed by undamaged bases, in addition to recognizing damaged bases (88). Future in vitro studies with the highly purified components of the NER system may provide further insights into the substrate requirements.

    ACKNOWLEDGEMENTS

We thank Drs. Gregory M. Landes for advice; John Bouck (Baylor College of Medicine) for computer searches of the human genome; Nora Goosen (Leiden Institute of Chemistry, The Netherlands) and Benedicte Michel (Institut National de la Recherche Agronomique, France) for E. coli strains; and E. Lynn Zechiedrich (Baylor College of Medicine), Gregory G. Germino (The Johns Hopkins University), Ravi R. Iyer, and Richard R. Sinden for critically reading the manuscript, encouragement, and advice.

    FOOTNOTES

* This work was supported by Polycystic Kidney Research Foundation Grant 98004, National Institutes of Health Grants GM 52982 and NS 37554, and the Robert A. Welch Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Center for Microbiology and Virology, Polish Academy of Sciences, 106 Lodowa St., 93-232 Lodz, Poland.

To whom correspondence should be addressed: Institute of Biosciences and Technology, Center for Genome Research, Texas A & M University System Health Science Center, Texas Medical Center, 2121 Holcombe Blvd., Houston, TX 77030-3303. Tel.: 713-677-7651; Fax: 713-677-7689; E-mail: rwells@ibt.tamu.edu.

Published, JBC Papers in Press, February 27, 2001, DOI 10.1074/jbc.M100845200

    ABBREVIATIONS

The abbreviations used are: PKD, polycystic kidney disease; kbp, kilobase pair; NER, nucleotide excision repair; ADPKD, autosomal dominant polycystic kidney disease; CFU, colony-forming units; bp, base pairs; Ap, ampicillin.

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