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
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ABSTRACT |
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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.
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.
E. coli Strains
JTT1 ((gal-25, 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 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 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 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,
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 + 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).
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 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.
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.
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.
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
Third, we investigated the E. coli SOS strains. During the
preparation of JJC523 (
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
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
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
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
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
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
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
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
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.
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
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, 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 (
uvrA::cam), CS5429:KMBL1001
(
uvrB::cam), CS5430:KMBL1001
(
uvrC::cam), and CS5431:KMBL1001
(
uvrD::tet) (24) were obtained from
Dr. Nora Goosen (Leiden Institute of Chemistry, The Netherlands). Strains JJC123 (GY6781;
[sfiA::lacZ],
pro-lac, gal+,
rpsL, mal::Tn9, LexAind1), JJC510
(GY4786;
[p(sfiA::lacZ) CIind
]
lac-pro,
rpsL), and JJC523 (GY5425; recA441, sulA
II,
lacI169, thi, leuB6,
his4, argE3, ilvTS, galK2,
rpsL37, lexA71::Tn5) were kind gifts of Dr. Benedicte Michel (Institut National de la Recherche Agronomique, France).
(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.).
10 mg/ml, and
their concentrations were determined spectrophotometrically from their
extinction coefficients (
307 = 6 × 103
(25),
293 = 2.02 × 104 (26), and
244 = 2.81 × 104 (25)), respectively.
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 standard error of t2
(Et2) was derived from the standard
error of k (Ek) according to
Et2 = t2Ek/k.
(Eq. 1)
t, and
t is the number of ligand-induced superhelical turns. For an in vivo
population of negatively supercoiled topological isomers,
<Lk> = Tw0 + <Wr> (<Wr> = Wr0 + <
>, where
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
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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.
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.
View larger version (22K):
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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.
E. coli strains
Parameters for the growth curves
uvrB and
uvrC strains
compared with their isogenic wild-type KMBL1001. In addition, for
uvrB there was no lengthening of the doubling time,
whereas
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
uvrA and
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.
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.
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.
View larger version (20K):
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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.
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.
View larger version (27K):
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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 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
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.
30
µM actinomycin D, supporting the hypothesis that the
formation of underwound non-B DNA structures was responsible for cell loss.
uvrB carrying pBS4.0. Contrary to the wild-type strain,
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.
uvrB and
uvrC mutants (the
uvrA and
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.
View larger version (26K):
[in a new window]
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,
uvrB,
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,
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
uvrB.
lexA71 and lexAind1 strains, respectively, did
not affect the retention of pSPL3.
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
lexA71 cells were difficult to interpret because of the
substantial lysis observed in the presence of pBS4.0, as noted above.
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.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
uvrA and
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).
View larger version (21K):
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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.
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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.
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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.
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
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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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