©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Tandem Sequence Duplications Functionally Complement Deletions in the D1 Protein of Photosystem II (*)

Hadar Kless (§) , Wim Vermaas

From the (1)Department of Botany and Center for the Study of Early Events in Photosynthesis, Arizona State University, Tempe, Arizona 85287-1601

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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, 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.


INTRODUCTION

Photosystem II (PS II)()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) .

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 (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.


EXPERIMENTAL PROCEDURES

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.

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) .

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 µEms.

Measurements to determine the rate of oxygen evolution at saturating light conditions, the relative variable fluorescence (the ratio of variable and maximal fluorescence, F/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.


RESULTS

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.

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.


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 4.810 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.

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 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.

The deletion mutant 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 ().

YR1-3, the three pseudorevertants originating from 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.

Several fluorescence parameters also were modified at an elevated temperature. After 10 min at 37 °C, F (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 ().


DISCUSSION

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).

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 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.

The deletion in the 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.

The NR4 pseudorevertant isolated from the 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.

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 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.

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.

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.

The frequency with which photoautotrophic colonies were obtained was about 210 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 0.2-810 per cell(21) , which may suggest a common molecular basis for sequence duplication in these bacteria.

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.

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.

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.

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.

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).

  
Table: Photosynthetic performance of wild type and the various mutants

Strains that were analyzed are wild type (WT) and the second-site revertants that originated from the deletion 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 Omg Chlh). 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 tvalues 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

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 = F - F was measured after 10 min of incubation at 37 °C and is normalized to that measured at room temperature.



FOOTNOTES

*
This research is supported by National Institutes of Health Grant 1R01GM51556. This is publication No. 236 from the Arizona State University Center for the Study of Early Events in Photosynthesis. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 602-965-3698; Fax: 602-965-6899.

The abbreviations used are: PS II, photosystem II; PCR, polymerase chain reaction; Q, the second electron-accepting plastoquinone in PS II; µE, microeinsteins; DCMU, (3[3,4-dichlorophenyl]-1,1-(dimethylurea)).


ACKNOWLEDGEMENTS

We thank Julia Prescott for excellent technical assistance and Dr. Svetlana Ermakova-Gerdes for careful review of the manuscript.


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