From the
Obligate photoheterotrophic mutants of the cyanobacterium Synechocystis sp. PCC 6803 that carry deletions of conserved
residues in the plastoquinone-binding niche of the D1 protein were used
to select for spontaneous mutations that restore photoautotrophic
growth. Spontaneous pseudorevertants emerged from two deletion mutants,
Photosystem II (PS II)
Several mutants of the cyanobacterium Synechocystis sp. PCC
6803 that contain such deletions of highly conserved residues in the D-E region of the D1 protein were unable to grow
photoautotrophically(11) . We aimed to study whether spontaneous
changes can occur that lead to functional complementation of the
deleted residues in the D1 protein in these mutants and to restoration
of photoautotrophic function. Several spontaneous second-site
revertants were isolated from two of these strains
(
To select for restoration of photoautotrophic growth,
a 100-ml cell culture was grown in the presence of 5 mM glucose (which is required for photoheterotrophic growth) and was
harvested in late logarithmic phase and concentrated by centrifugation
(2500 rpm for 10 min at room temperature). Cells were resuspended
(final density of about 1 OD at 730 nm) in 500 ml of BG-11 medium
supplemented with 1, 2, and 5 mM glucose for further growth.
At various time points during growth, samples of 50 ml were taken out
of the liquid cultures, concentrated to 1 ml, and spread on BG-11
plates without glucose and containing 20 µg/ml spectinomycin, 2.5
µg/ml chloramphenicol, and 10 µg/ml kanamycin. Genes conferring
resistance against these antibiotics were used to delete parts of psbAI and psbAIII (16) and to introduce the
deletion mutation in psbAII(11) .
Measurements to determine the rate of oxygen evolution at saturating
light conditions, the relative variable fluorescence (the ratio of
variable and maximal fluorescence, F
The conditions
that might favor such spontaneous reversion were studied further in
these mutants by varying the initial glucose concentration in the
liquid medium and the time of growth under non-selective conditions. In
several attempts to isolate revertants (exemplified in Fig. 1),
the appearance of photoautotrophic strains seemed to be restricted to
growth phases in which growth is suppressed. As the plating efficiency
of photoautotrophic colonies under our experimental conditions was
about 10-20% (not shown), the spontaneous reversion event seemed
to occur at the very end of the logarithmic phase or during the
stationary phase.
Among the various experiments, photoautotrophic colonies emerged
more often (about 3-fold) in cultures that were started in the presence
of 1 or 2 mM glucose than if 5 mM was present in the
medium (not shown). The presence of lower initial glucose
concentrations appeared to increase the frequency with which
second-site revertants occurred and established. This may be related to
the higher cell density accumulated in the presence of higher glucose
concentrations and the smaller competitive advantage of photoautotrophy
at this stage.
The deletion mutant
YR1-3, the three pseudorevertants originating from
Several fluorescence parameters also were modified
at an elevated temperature. After 10 min at 37 °C, F
The photoautotrophic capacity of some of the mutants
isolated in this study may be rather surprising. In the three YR
pseudorevertants, an in-frame duplication of DNA coding for part or all
of the putative de helix has occurred; the de helix
is a part of the Q
The deletion in the
The NR4 pseudorevertant isolated from
the
Some of the
duplication mutants exhibited an unusual sensitivity to increased
temperature. This may reflect an increased tendency to thermal
denaturation of protein interactions in the Q
In all pseudorevertants, the deletions in the
protein were corrected by insertions that extended the region to a size
of the D1 protein equaling or exceeding that in wild type. Since
different compositions and lengths of the protein sequence could be
functionally accommodated in this region, it seems that a minimal
length of the protein sequence is required in these regions, perhaps to
serve as a spacer linking two neighboring sequences that interact with
different, spatially distinct components.
The frequency with which photoautotrophic colonies were
obtained was about 2
Different types of DNA
modifications could be involved in spontaneous DNA modifications in Synechocystis 6803. For instance, corrections of a frameshift
mutation in the psbDI gene coding for the D2 protein were
found to include deletion, insertions, and tandem sequence
duplication(17) . In the current study, particular DNA
duplications were selected as a solution that functionally complemented
the deletions merely because other duplications, further deletions,
point mutations, out of frame insertions, and other possible
modifications failed to result in a functional PS II. This may
furthermore suggest that in-frame duplication of tandem sequences is a
relatively common way for Synechocystis 6803 to introduce
additional DNA sequences into a gene.
Two
main mechanisms have been postulated to yield sequence duplication: 1)
slippage synthesis of misaligned sequences and 2) unequal exchange
between identical
sequences(24, 25, 26, 27) . In the first
mechanism, synthesis of misaligned sequences requires direct repeats
near the borders of the duplication that allow homologous pairing
between the polymerized and the template strands. One of the repeats of
the polymerized strand is misaligned to the other repeat in the
template, and the DNA sequence between these repeats forms an unpaired
loop, which eventually results in a tandem insertion in the polymerized
strand. In the latter mechanism, unequal exchange or crossover may
occur between identical regions in gene copies or similar loci of a
chromosome pair. First, two similar regions of two separate molecules
are misaligned by homologous pairing to form a heteroduplex; then,
strand invasion and unequal reciprocal recombination occurs, resulting
in one chromosome with a tandem duplication and the other with a
deletion.
Both mechanisms require sequence homology to allow strand
misalignment that initiates the duplication process. However, in all
six pseudorevertants, the inserted DNA pieces do not seem to have
sufficient homologous sequences that can be aligned by Watson-Crick
base pairing at either of the two possible insertion locations (at the
3`- or 5`-ends of the duplication) ( Fig. 3and Fig. 4).
Thus, the formation of misaligned sequences is unlikely to be involved
in the observed duplications. The absence of homologous sequences and
clear secondary structure near tandem duplications has been reported
for other systems (21), but to our knowledge no mechanism leading to
such duplications has been proposed yet.
Obviously, the proposed
mechanism is very flexible in terms of the location and size of the
duplication. This flexibility, which is evident in the YR and NR
pseudorevertants, may be an important asset in evolutionary terms as it
allows the cell population to check, on a trial and error basis,
different possibilities to modify genes by partial sequence
duplication. At the protein level, such duplications may result in the
introduction of novel sequences (as is the case in NR2).
Strains that were analyzed are wild type (WT) and
the second-site revertants that originated from the deletion mutants
On-line formulae not verified for accuracy ) was estimated from flash-induced
fluorescence measurements. This constant was calculated from the
fluorescence level 23 ms after the flash (12). Thermosensitivity (ts)
indicates the ability (-) or inability (+) to grow
photoautotrophically at 37 °C; ± indicates poor but
noticeable growth. The effect of temperature on the variable
fluorescence F
We thank Julia Prescott for excellent technical
assistance and Dr. Svetlana Ermakova-Gerdes for careful review of the
manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
YNIV
and
NN
, when
the cultures were maintained long after the carbon source (glucose) had
been depleted from the medium and cells had reached stationary phase.
Most pseudorevertants were found to contain tandem duplications of
6-45-base pair DNA sequences located close to the domain carrying
the deletion; none of them restored the wild-type sequence. Three
pseudorevertants isolated from the
YNIV
mutant contained a duplication (7-15 codons) of the DNA
sequence immediately downstream of the deletion; the protein region
encoded by this DNA may include part of the putative de helix,
an important constituent of the plastoquinone-binding niche. Three
pseudorevertants isolated from the
NN
mutant
contained duplications corresponding to 2-8 amino acid residues
adjacent to the site of the deletion. In all six pseudorevertants
carrying duplications, the length of the D1 protein in the modified
regions was restored to at least the length present in wild type,
suggesting that a minimal length of these protein domains may be
required for functional integrity. In another photoautotrophic strain
isolated from
NN
, no secondary mutations
could be identified in the gene coding for the D1 protein; such
mutations apparently reside on another protein subunit of the
photosystem II complex. Photosystem II function in the pseudorevertants
was altered as compared with wild type in terms of growth and oxygen
evolution rates, photosystem II concentration, the semiquinone
equilibrium at the acceptor side, and thermostability. A mechanism
leading to tandem sequence duplication may involve DNA damage followed
by DNA synthesis, strand displacement, and ligation.
(
)is a large
protein complex that is located in the thylakoid membranes of plants,
algae, and cyanobacteria and utilizes light energy for plastoquinone
reduction by water(1) . The arrangement of central protein
subunits, prosthetic groups, and cofactors involved in PS II function
has been modeled based on detailed structures available for comparable
reaction centers of purple bacteria(2, 3) . One of the
most extensively studied protein subunits of PS II, the D1
protein(4) , is highly conserved phylogenetically (5) mainly in regions thought to be of functional
importance(6) . A particular domain of interest in the D1
protein is the D-E region, which is located between
the fourth and fifth (D and E) transmembrane helices
(7) and contains residues that contribute to a binding niche that can
accommodate the secondary quinone Q
and several PS II
inhibitors. Many conserved residues in the D-E region were
shown to be functionally replaceable and may reside in a domain that is
flexible in terms of its primary sequence. On the other hand, several
deletion and replacement mutations of other conserved residues in the D-E region, particularly those located near the putative de helix, led to a loss of photoautotrophy, possibly by
disrupting interactions with Q
that are crucial for PS II
activity(8, 9, 10, 11, 12) .
YNIV
and
NN
) and
were found to contain tandem sequence duplications near the location of
the deletions. We studied the conditions needed to isolate second-site
revertants and analyzed the effects of these mutations on PS II
properties.
Strains and Growth Conditions
The
cyanobacterium Synechocystis sp. PCC 6803 used for this study
is naturally transformable and undergoes very efficient homologous
double recombination facilitating targeted gene
replacement(13, 14) . This strain can also grow
photoheterotrophically in the presence of glucose as an alternative
carbon source; this allows the isolation of mutants impaired in PS II
function. Synechocystis 6803 wild-type and mutant cultures
were grown on plates or in liquid in BG-11 medium (15) as
described(12) . 5 mM glucose and 20 µM atrazine were included in the BG-11 plates used to maintain the
strains. Glucose (5 mM) was included also in liquid cultures
of cells grown for oxygen evolution and fluorescence measurements. The
obligate photoheterotrophic mutants with the deletions in the D1
protein, YNIV
and
NN
, have been described(11) . The
nomenclature of these strains correlates with the residues that have
been deleted.
Glucose Assay
Glucose concentrations in
liquid medium were followed during cell growth using a colorimetric
assay with o-toluidine reagent (Sigma). 100 µl of medium
and 5 ml of the reagent were mixed and boiled for 10 min in a water
bath. Glucose concentrations were determined by measuring the
absorption at 635 nm and by comparing the calorimetric reaction of the
samples taken from the growth medium with those of known glucose
standards (0.05-10 mM).
PCR and DNA Sequencing
Isolation of
genomic DNA from the various mutants, PCR amplification of the psbAII gene, and sequencing of the PCR products were performed
as described (12). The sequence (5` 3`) of the primers used for
PCR amplification of the psbAII gene was CCAAAACGCCCTCTGTTTACC
(A) and GGATTAATTCTCTAGACTCTCTAATGG (B). For each of the six
pseudorevertants with secondary mutations in D1, the region for which
the sequence was determined included codons 223-278 of the psbAII gene.
Complementation Assay
To determine the
location of the secondary mutation that restored photoautotrophy, a
complementation assay was performed. The psbAII gene was PCR
amplified from the genome of the various strains with restored
photoautotrophic capacity using primers A and B. The PCR products were
precipitated, and 1 µg was used to transform the corresponding
deletion mutants (YNIV
or
NN
) followed by selection for
photoautotrophic phenotype on plates in the absence of glucose as
described(17) .
Analysis of Photosynthetic
Performance
Photoautotrophic growth rates were measured by
turbidity (optical density at 730 nm, OD < 0.5) using liquid
cultures (BG-11, 50 ml) shaken at 100 rpm on a rotary shaker at 30
°C and at a light intensity of 50-60
µEm
s
.
/F
),
the kinetics of fluorescence decay (t
of two
decay phases), and estimation of K
, the
apparent equilibrium constant between the semiquinones at the acceptor
side (Q
Q
and Q
Q
), were carried out as
described(12) . The concentration of PS II centers on a
chlorophyll basis was determined from a competitive binding assay of
C-labeled DCMU using intact cells as
described(18) .
Thermosensitivity
Thermosensitivity of
photoautotrophic growth was assayed on BG-11 plates in the absence of
glucose. Equal amounts of wild-type and pseudorevertant cells were
grown on plates incubated at 30, 34, and 37 °C, and the relative
growth was estimated visually after 10 days. The effect of temperature
on the variable fluorescence (F = (F
- F
)/F
) was followed by monitoring
the flash-induced fluorescence (12) after cells were incubated
at 37 °C in the dark for 10 min.
Conditions for Spontaneous
Reversion
Several mutants of Synechocystis 6803
carrying 2-4 residue deletions in the D-E region of the
D1 protein were impaired in PS II activity and could not grow
photoautotrophically(11) . These mutants failed to yield
spontaneous photoautotrophic pseudorevertants when cells were grown
under optimal growth conditions in the presence of glucose and
harvested for selection before or at the end of the logarithmic phase
(not shown). However, such second-site revertants were obtained from
two obligate photoheterotrophic mutants, YNIV
and
NN
, when the cultures were
maintained until long after all glucose had been taken up from the
medium and cells had entered the stationary phase.
Figure 1:
Isolation of pseudorevertants from a
deletion mutant of D1. Growth curves (turbidity at 730 nm, opensymbols) and number of pseudorevertants (colonies grown
on plates without glucose per 50 ml of culture, closedsymbols) were measured for the deletion mutant
NN
as a function of time. At time 0,
cultures were diluted at the same initial turbidity in medium
containing 5 (squares), 2 (triangles), or 1 (circles) mM glucose. The remaining amounts of
glucose in the media were followed during these experiments. 1 OD at
730 nm corresponds to about 4.8
10
cells/ml.
Photoautotrophic colonies were isolated from
cultures initiated with three different glucose concentrations (Fig. 1). The colonies isolated from each culture (about
5-10) were found to contain the same DNA sequence, suggesting
that they originated from the same parent cell that had acquired a
spontaneous second-site mutation. Thus, the appearance of
pseudorevertant cells as a function of time in Fig. 1is a growth
curve of photoautotrophic cells originating from a single parent.
The Molecular Basis of Second-site
Mutations
The protein regions carrying the deletion
mutations may be crucial for the binding of Q and for
electron and proton transfer involving Q
at the reducing
side of PS II (Fig. 2). Therefore, (pseudo) revertants generated
from these mutants are expected to carry (secondary) mutations near the
Q
-binding environment. The locus of the spontaneous
mutations in the pseudorevertants was determined by a functional
complementation assay. In most pseudorevertants, the complementation
assay indicated that the secondary mutation was located in the psbAII gene. However, in a few cases involving
photoautotrophic mutants originating from the
NN
deletion mutant (e.g. NR4), the complementing change was
not found to be in the psbAII gene.
Figure 2:
The region of the wild-type D1 protein
between the transmembrane helices D and E. The D-E region is modeled based on the proposal by Trebst (7),
indicating the proposed arrangement of the transmembrane helices D and E, the putative de helix, and the loop
regions D-de and de-E in between. Residues that were
shown to be important for PS II function (11) were positioned closer to
the membrane surface and to where Q (large Q) is
thought to be bound (see also Ref. 31). The residues that were deleted
in the
YNIV
and
NN
mutants are shaded.
The PCR fragments
amplified from the psbAII gene of the various photoautotrophic
pseudorevertants were sequenced for at least 80 nucleotides at both
sides of the deletion. A total of six independent second-site
revertants with different psbAII gene sequences were detected.
All pseudorevertants contained in-frame duplications of tandem
sequences near the deletion mutation. True revertants that restored the
wild-type sequence were not identified.
YNIV
lacks four residues in the D-de loop of the D1 protein (Fig. 2) and has an extra Glu residue
inserted at position 245 (Fig. 3). The three pseudorevertants
isolated from this mutant were found to contain duplications of nearby
DNA pieces corresponding to 7-15 codons, virtually duplicating at
least part of the putative de helix of D1 (Fig. 3). As
compared with wild type, the protein sequence in this region was
extended by 4-12 residues. The 5` location of the duplicated
piece is identical in all three pseudorevertants, suggesting that a
similar mechanism was involved at this end of the duplication.
Figure 3:
Nucleotide and amino acid sequence between
residues 242 and 270 of the D-E region of the D1 protein in
wild-type Synechocystis sp. PCC 6803 (WT), the
YNIV
deletion mutant, and three second-site
revertants (YR1-3). The
YNIV
mutant
carries a Glu residue (underlined) inserted between residues
244 and 245 (11). Residues of the putative de helix are italicized. In all of the revertants, a region of the psbAII gene (underlined) located downstream of the
deletion was duplicated leading to a 7-15 codon insertion. The
inserted pieces are aligned below the original sequence and could have
been inserted either at the 3`- or 5`-end of the duplicated sequence,
resulting in the same end product.
From
the other mutant, NN
, missing two residues
in the loop between helices de and E (Fig. 2),
four pseudorevertants were isolated. Of these, three different
pseudorevertants were found to carry duplication of 2-8 codons
copied from a flanking region and inserted at the deletion location (Fig. 4). In two of these pseudorevertants, NR1 and NR2, the
length of the D1 sequence was restored to that in wild type, while in
NR3 this region was extended by 6 codons as compared with wild type.
For the fourth pseudorevertant, NR4, in which the complementation assay
indicated that the second-site mutation was not in the psbAII
gene, the sequence of the D1 protein in this region was identical to
that in the original
NN
mutant.
Figure 4:
Nucleotide and amino acid sequence between
residues 257 and 273 of the D-E region of the D1 protein in
wild-type Synechocystis sp. PCC 6803 (WT), the
NN
deletion mutant, and three second-site
revertants (NR1-3). The obligate photoheterotrophic
NN
mutant lacks two amino acids essential
for photoautotrophy, and has an Arg-269 codon with a silent mutation (underlined) (11). Residues of the putative de helix
are italicized. The isolated second-site revertants have
duplications of 6-24 nucleotides (underlined) that
include the deleted location in the psbAII gene. The inserted
pieces are aligned below the original sequence and could have been
inserted either at the 3`- or 5`-end of the duplicated
sequence.
Functional Consequences of the Sequence Duplications
on PS II Activities
The spontaneous duplications restored
photoautotrophy but modified the protein sequence near the Q niche. PS II properties related to Q
were measured in
wild type and the second-site revertants ().
YNIV
, appeared to contain a decreased amount
of PS II and were found to have slow Q
oxidation by Q
(, Fig. 5). The
relative variable fluorescence, F
/F
, was
less affected. The half time of the fast phase of fluorescence decay
was about 3-fold longer, and that of the slow phase was increased by
about 7-18-fold, suggesting an overall decrease in the rate of
electron transfer between Q
and Q
in these
mutants. In all of the mutants, the amplitude of the fast phase of
fluorescence decay was about 60-80% of the total fluorescence;
the remainder decayed at a slower rate. The apparent semiquinone
equilibrium constant K
in the
pseudorevertants was decreased by more than 1 order of magnitude as
compared with wild type, consistent with a destabilization of
Q
. It should be noted that the value of K
is governed by factors including
Q
affinity and Q
stability
and protonation, which affect the occupancy of the Q
niche
(12).
Figure 5:
Decay kinetics of flash-induced variable
fluorescence in intact cells of wild type (WT) and two
pseudorevertants, YR1 and NR1. A single turnover flash was given at t = 0; the fluorescence decay was followed for 23 ms
after the flash (12). Each curve represents an average of 10
measurements and is normalized by (F - F)/F). The
initial fluorescence F was set to
zero.
The four NR1-4 pseudorevertants originating from
NN
were somewhat less impaired than the
YR1-3 pseudorevertants in terms of Q
oxidation, the slow phase of the fluorescence decay, and the
semiquinone equilibrium (, Fig. 5). However, the NR
mutants had a lower F
/F
ratio and less PS
II on a chlorophyll basis, suggesting that PS II reaction centers were
less stable. The NR4 pseudorevertant, in which the location of the
complementary change was not identified, had a rather normal
semiquinone equilibrium constant (>20). This distinguished NR4 from
the other pseudorevertants.
PS II Thermostability
Point mutations and
length modifications in a conserved region of the D1 protein might
affect the thermostability of functional PS II reaction centers.
Possible changes in thermostability as compared with wild type were
analyzed by measuring photoautotrophic growth and variable fluorescence
at an elevated temperature. We defined strains to be thermosensitive if
they could not grow photoautotrophically at 37 °C ().
The differences between temperature-sensitive and resistant strains
were more clearly distinguished at this temperature, although
thermosensitive mutants grew noticeably slower at 34 °C than at 30
°C as well.
(the basal fluorescence) and F
(in this case the maximal flash-induced
fluorescence in the absence of DCMU) were increased in all strains.
However, the variable fluorescence at 37 °C, F
= F
- F
, was dramatically
reduced (by about 40-80%) in the thermosensitive mutants (YR3 and
the NR strains), while in wild type and in YR1 and YR2 it was hardly
affected ().
Structure-Function Relationship in the Modified
Protein Regions
The D-E region of the D1 protein
is highly conserved phylogenetically(5) . Deletion of amino
acids 246-249, 254-257, 266-267, or 268-270 in
the D-E region was found to lead to a loss of PS II function,
photoautotrophy, and oxygen evolution, indicating the relative
importance of these domains in reducing side activities of PS
II(11) . In this study, pseudorevertants were isolated from the
mutants lacking residues 246-249 and 266-267. Using the
same conditions to isolate spontaneous pseudorevertants, we were not
able to obtain any photoautotrophic colonies from the deletion mutants
lacking residues 254-257 or 268-270 of D1 (however, see
Ref. 12).
niche(7) , and it shows an
amphiphilic arrangement of amino acids. In YR2 and YR3 mutants, the de helix appears to have been duplicated as a whole, yielding
two identical parts that could be functionally involved in the
Q
-binding niche. In the YR1 and YR2 pseudorevertants, which
were less impaired functionally than YR3, 7 and 14 residues were
inserted in the putative helical region, respectively. These insertions
correspond to approximately two and four additional turns of the helix,
respectively, which may maintain amphiphilicity and overall orientation
of this domain. On the other hand, insertion of 15 residues as in YR3
would disrupt the amphiphilic nature of the helix. Indeed, in a
functional sense, YR3 is more impaired and thermosensitive as compared
with YR1 and YR2, which is what would be expected if the duplications
became part of an extended helix.
NN
mutant was functionally restored in the
three pseudorevertants (NR1-3) by duplications of 6-24
nucleotides that either restored the D1 protein to its original length
(NR1 and NR2) or extended it by six amino acids (NR3). These mutants
have an altered K
as compared with wild
type, indicating involvement of the 266-267 region in determining
the properties of Q
. Some properties of the NR
pseudorevertants, most clearly the rate of the slow component of
fluorescence decay (t
, ), are
distinctly different from those of the YR strains (and are quite
similar to those of wild type), suggesting that the regions
246-249 and 266-267 constitute distinct parts of the
Q
-binding niche.
NN
mutant did not carry a complementary
change in the psbAII gene coding for the D1 protein and was
found to have the
NN
sequence near the
deletion. In contrast to both the NR1-3 and YR pseudorevertants,
NR4 has a rather normal K
. It is unlikely
that such a deletion mutation can be complemented as a result of, for
example, tRNA suppressor mutations. Another protein subunit of the PS
II complex, probably one that is close to the Q
site of the
D1 protein, may carry the second-site mutation. However, according to
complementation experiments, the D2 protein, which is known to be in
close association with D1 in the reaction center of PS II, was not
found to carry the secondary mutation in this case.
niche in
these mutants. This may also suggest that mutants with less stable PS
II centers resulting in reduced PS II activity may need to be grown and
handled at lower (<30 °C) temperatures to prevent potential
thermal damage.
Spontaneous Complementation by Second-site
Mutations
Certain physiological features of the
cyanobacterial culture under the conditions applied in this study
seemed to induce a higher frequency of tandem duplications resulting in
photoautotrophic pseudorevertants. Complementation of a missing
functional trait by a second-site mutation within the impaired gene may
be comparable to ``adaptive mutations'' as described in Escherichia coli and yeast(19) , although there is no
evidence in our case (or in other cases, for that matter) that the
duplication events occurred preferentially in the gene carrying the
deletion mutations. However, as in adaptive mutations(20) , the
mutational events reported here are detected mainly under adverse
conditions.
10
per cell as estimated
from the amount of cells in the cultures when the first pseudorevertant
was generated (extrapolated from the pseudorevertants growth curve) (Fig. 1). This frequency is lower than that of spontaneous
mutations of a single nucleotide (C to T or G to A) change, which is
about 10
per cell in E. coli and about
10
-10
per cell in
cyanobacteria (not shown). The duplication frequency is comparable with
that noted for duplication of plasmid sequences in E. coli (about 0.2-8
10
per
cell(21) , which may suggest a common molecular basis for
sequence duplication in these bacteria.
Mechanisms of Tandem Sequence
Duplication
Repetitive DNA sequences and gene families that
may have originated by tandem sequence duplications are widespread in
many organisms(22, 23) . These sequences seem to have a
role in genome organization and stabilization. However, the mechanisms
that may lead to sequence duplications are not yet understood.
A Postulated Mechanism for Tandem Sequence
Duplication
A possible mechanism for sequence duplication
in the absence of homologous pairing near the duplication borders is
illustrated in Fig. 6. First, one of the DNA strands is nicked;
then, the free 3`-OH group generated by the nick is used for extension
by DNA polymerase that displaces the complementary strand; the
displaced strand folds back, and its 5`-end is ligated to the 3`-end of
the newly extended strand; eventually, the strand carrying the
duplication is copied by mismatch repair enzymes. This mechanism is
relatively simple and requires only a few steps that can be performed
by enzymes involved in DNA repair and recombination. The duplication by
this mechanism basically has no size limitation and can explain the
occurrence of duplications in any DNA sequence.
Figure 6:
Model
for the formation of a tandem sequence duplication. A nick in the DNA
serves as a priming site for DNA polymerization that displaces the
``old'' strand. The displaced strand then folds back and
religates to the end of the extended strand (see text for more
details).
The borders (and
size) of the duplications may be determined, at one end, by a nick that
primes DNA polymerization and strand displacement (and that may also
attract DNA repair enzymes). At the other end, in the absence of clear
Watson-Crick homology, other means such as triplex recognition (28, 29) and proteins of DNA repair complexes (30) may stabilize the DNA ends for ligation. The relative rate
of DNA polymerization versus ligation may also play a role in
determining the duplication boundaries.
Table: Photosynthetic performance of wild type and the
various mutants
YNIV
(YR1-3) and
NN
(NR1-4). In the two deletion
mutants, no photoautotrophic growth, oxygen evolution, and variable
fluorescence are observed (11). Growth rates are indicated by the
doubling time in hours measured in liquid cultures under
photoautotrophic conditions (without glucose). Oxygen evolution rates
are given relative to those of wild type (wild-type rates are 220
± 20 µmol O
mg
Chl
h
). The relative
variable fluorescence F
/F
was
measured at the same chlorophyll concentration and is normalized to
that of wild type (about 0.6). The amount of PS II centers on a
chlorophyll basis (molar ratio of PS II/chlorophyll) is normalized to
that of wild type, where this ratio is about 1/680. The t
and t
values are the half time (in
ms) of the fast and slow decay components, respectively, of the
flash-induced fluorescence decay (Fig. 5). The apparent semiquinone
equilibrium constant K
= F
- F
was measured after 10 min of incubation at 37 °C and is
normalized to that measured at room temperature.
, the second electron-accepting plastoquinone in
PS II; µE, microeinsteins; DCMU,
(3[3,4-dichlorophenyl]-1,1-(dimethylurea)).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.