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Molecular Mechanisms of TRS Instability

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Introduction

Microsatellites, stretches of short, tandemly repeated motifs of one to six nucleotides are very unstable and display very high polymorphism among individuals.14 Of these repeats, a special class of microsatellites, trinucleotide repeat sequences (TRS), are involved in human neurodegenerative diseases.5,6 To date, 14 neurological or neuromuscular hereditary human disorders, also called mental retardation diseases, have been linked to the genetic instability of the TRS. Diseases including myotonic dystrophy, Huntington's disease, Kennedy's disease, fragile X syndrome, spinocerebellar ataxias or Friedreich's ataxia result from expansion of trinucleotide sequences such as (CTG/CAG)n, (CGG/CCG)n, or (GAA/TTC)n present in human genome.7

The unstable TRS may expand and thus, depending on their localization in the chromosomes, may disturb an expression of crucial genes. The function of the majority of genes which activity is affected remains unknown. It is clear, however, that common features of the diseases mentioned above result as a consequence of the expansion of the TRS. Moreover, the inheritance of such diseases cannot be explained by Mendelian genetics due to the character of expansions described as dynamic mutations.8,9 Mutations of this type result in a length change of DNA tracts containing repeated sequences. From the clinical point of view, the increasing length of the same TRS region causes a progressive increase in expressivity of the mutation over a number of generations. Such phenomenon is termed “anticipation”.10 There is an inverse relationship between the age of onset and the size of repeat and a direct relationship between expansion size and disease severity. The length of expanded TRS in afflicted families may vary from as much as tens of triplet repeats (Huntington's disease) to a few thousand repeats (myotonic dystrophy).

Over a decade of an extensive study on the nature of the genetic instabilities of TRS revealed that size alterations of these tracts may be generated via different biochemical mechanisms, including replication, transcription, DNA repair and recombination. Additionally, experiments in bacteria, yeast and mammalian systems suggest that an elevated frequency of length changes (expansions and deletions) of TRS is caused by their propensity to form unusual secondary structures.11

It is interesting that each of the neurodegenerative disorders mentioned above shows a highly defined threshold for the TRS tract length (usually 30–40 repeats, specific for each disease) beyond which the instability of such sequences increases dramatically, leading to the massive expansions. It is not known what drives short trinucleotide tracts present in healthy individuals to reach such threshold level. One plausible explanation of the origin of TRS-related disorders assumes a slow accumulation of small-increment length changes (SILC) which eventually pushes a repeated sequence to reach the threshold level. Beyond this level, further massive length changes (MLC) seem to be inevitable. One might assume that SILC and MLC are due to the character of DNA containing triplet repeats of different lengths (below or above the threshold) that may serve as distinct substrates for cellular factors. It cannot be excluded that maintaining the number of the repeats at the safe level results from the subtle balance between the rate of expansions versus deletions.

In this chapter, we emphasize the role of the non-B-DNA conformations as a primary source of the genetic instability of TRS. Depending on the length, type of tracts or the presence of interruptions, the character of repeated sequences creates diverse opportunity for such alternative secondary DNA structures formation. Their presence may have significant impact on the course of the metabolic processes like replication, transcription, repair and recombination occurring in the given DNA region, leading in effect to the different modes of instability.

Secondary DNA Structures as a Source of TRS Instability

It has been known for some time that microsatellites can differ in repeat number among individuals and influence the integrity of genetic information. Alterations in the size (insertions and deletions) of DNA are not limited to the TRS-dependent disorders, as other microsatellite instabilities are also observed in tumors from patients with hereditary nonpolyposis colorectal cancer (HNPCC).1216

Several factors may contribute to the mutational dynamics of microsatellite DNA, including number of repeats, composition and length of the repeating motif, presence of interruptions within the sequence and the rate of intracellular processes such as replication, transcription, repair, or recombination.17 Experiments in Escherichia coli demonstrated that such sequences may gain or loose repeats. Most of the early work demonstrated that the instability did not depend on a RecA function of a host strain, suggesting that recombination was not the predominant mechanism generating microsatellite variability.18 A significant feature of the direct (tandem) repeats is their intrinsic ability to form non-B-DNA conformations.19 Unusual DNA structures (Fig. 1) such as left-handed Z-DNA, cruciforms, slipped-stranded DNA, triplexes, and tetraplexes may form due to their palindromic nature, under physiological conditions, also in vivo.2026 Such structures potentially may be hazardous for genome stability if not removed by repair mechanisms. Many experimental lines of evidence have shown that non-B-DNA-forming sequences are unstable and deleterious.

Figure 1. Non-B-DNA structures.

Figure 1

Non-B-DNA structures. Depending on the local base composition and the symmetry of a sequence, DNA may adopt various conformations including left-handed Z-DNA (alternating purine-pyrimidines such as d(GC)n), triplexes (polypurine/polypyrimidine tracts (more...)

Numerous in vitro studies have demonstrated the ability of the CTG/CAG and CGG/CCG tracts to form thermodynamically stable self-complementary hairpin structures and tetraplexes.2729 Hairpins assembled from CTG oligomers, as revealed by NMR study, form very stable antiparallel duplexes with TT pairs, whereas CAG oligonucleotides produce much less stable conformations which are destabilized by AA mispairs.30 This gives rise to unequal structural properties of repeated DNA during processes where single-stranded regions are involved, i.e., replication, transcription, repair or recombination. Hairpin structures will be formed and maintained more easily on the CTG strand than loops created on a strand containing CAG repeats. Similar studies confirmed that the Fragile X (CCG/CGG)ntriplets could also form hairpin structures although the (CCG)n strand more readily underwent self-pairing rearrangements than the complementary (CGG)n strand.31 In vitro measurements of the elastic constants of (CTG/CAG)n and (CGG/CCG)n and calculations of their free energy of supercoiling revealed their higher flexibility and their writhed structure in contrast with random DNA sequence.32,33 Interestingly, CTG/CAG and CGG/CCG repeats differ in their susceptibility to nucleosome formation. While former ones are prone to bind histone proteins, the latter generally prevent formation of the nucleosomes.3438 However, methylation level which determines the binding constant of the histones differs significantly between short and long CGG/CCG sequences as was shown by Godde et al.38 They pointed out that tracts shorter than 13 units upon methylation showed higher potential in nucleosome formation than long ones consisting of 74 repeats. Taken together, specific physicochemical features of these repeated sequences may be responsible for the alteration of the chromatin organization also in the neighboring regions.39 The flexible character of these TRS and their capability to form alternative DNA structures suggest they may act as a “sink” for the accumulation of superhelical density. Superhelical tension stabilizes secondary DNA structures and may be a crucial factor promoting the formation of structural abnormalities inside long TRS motifs. Non-B-DNA conformations may influence the activity of the enzymes involved in DNA processing.

It has been shown that many DNA polymerases pause within the long stretches of the (CTG/CAG)n and (CGG/CCG)n in vitro.40,41 The pausing sites of DNA synthesis at specific loci in the TRS depended on the length of the repeat tract and were abolished by heating at 70°C. These results suggest that appropriate lengths of triplet repeats adopt very stable non-B-DNA conformations that cause polymerases to pause during DNA synthesis. There is no direct evidence that polymerase stalling may produce expansions. However, such pausing may cause a primer-template realignment, which may lead to deletions and expansions, especially in repetitive sequences.

Formation of large hairpins, especially during the replication of the TRS, is believed to account for massive length alterations within these repetitive sequences, including expansion events linked to neurodegenerative disorders in humans. A novel type of instability based on duplications of CTG/CAG tracts (but not CGG/CCG motifs) including neighboring sequences were reported to frequently occur when cloned in R6K plasmids.42 Although this type of instability is not related to the neurodegenerative diseases, the presence of GAA/TTC and GAG/CTC tracts was probably responsible for the duplications in regions containing genes involved in development of neuroblastomas and malignant melanomas.43

Another structural aberration due to the slippage of complementary strands within the TRS is probably responsible for small deletions and expansions. Such a phenomenon was hypothesized to be the mechanism responsible for the slipped strand mispairing mutagenesis, the genetic hypermutability of dinucleotide repeat sequences in mismatch repair-deficient cells related to hereditary nonpolyposis colon cancer.1216,18,44,45As shown in an in vivo E. coli model utilizing methyl-directed mismatch repair (MMR) or nucleotide excision repair (NER) defective cells, long (CTG/CAG)n motifs cloned in plasmids exhibit very frequent length changes of 1–8 repeating units.46 These small expansions and deletions found in E. coli were studied in the absence of the repair functions since such activities would be expected to recognize and repair the looped structures formed within the slipped TRS conformation. The occurrence and the size of deletions and expansions present on plasmids isolated from single colonies were precisely monitored by the position of G to A interruptions present on the initial (CTG/CAG)n insert that served as valuable markers. The location of interruptions as compared to their original position indicated the type (expansion or deletion) and the size of the dynamic mutations observed in vivo.

Not only the (CTG/CAG)n tracts undergo dynamic mutations due to the slippage of the complementary strands. Similar analyses were also conducted on CGG/CCG fragile X and GAA/TTC Friedreich's ataxia sequences, where investigations showed that expansion and deletion products, differing in length from each other by one repeat or multiples of three base pairs could be resolved as distinct bands in polyacrylamide gel electrophoresis.46

Small-increment length changes (SILC) are believed to be a consequence of strand misalignment, leading to a formation of single-stranded loop-outs that consist of a few repeating units (Fig. 2). Such bubbles may be processed by endonucleolytic activities where excision of loops gives small deletions, whereas incisions opposite to loops produce expansions. The involvement of the nucleotide excision repair in SILC remains unclear and requires further investigations. The role of methyl-directed mismatch repair in generating genetic instabilities within the TRS will be discussed in next section.

Figure 2. Mechanism for SILC.

Figure 2

Mechanism for SILC. Following a denaturation and the formation of the slipped-DNA structure within the TRS, error-prone repair of resulting loop-outs produces small-increment expansions as a consequence of incisions opposite to loops and/or small-increment (more...)

In the case of long GAA/TTC stretches of more than 59 repeats, a novel, non-B-DNA structure has been detected in supercoiled plasmids in vitro and in vivo using a bacterial model.47 This structure is believed to originate from the self-association of two separate triplexes resulting in the formation of a very stable conformation termed “sticky DNA” (Fig.1). It has been shown that such structures may inhibit the process of DNA transcription and therefore could be responsible for the decreased amount of frataxin in FRDA patients.48,49 It does not explain though how GAA tracts may expand to reach the number of required repeats.

In the next sections we will discuss more specifically the possible influence of the secondary structures on the course of major DNA metabolism pathways.

Replication

Structural propensities of repeated DNA motifs, including the TRS, may cause such sequences to form slipped-stranded structures and hairpins during movement of the polymerase. In 1995 Kang et alfor the first time set up the in vivo (bacterial) system in which they were able to demonstrate that long (CTG/CAG)n tracts contained on ColE1 plasmids do undergo length changes after a number of cell generations.50 Although the instability pattern in E. coli shows strong bias toward deletions, this system provided also the evidence for expansion events. The great significance of this discovery was that it proved the usefulness of the bacterial system to study the genetic instability of human repeated sequences and enabled detailed analyses of the mechanisms of expansions that are the cause of many hereditary disorders. The observed instability strongly depended on the orientation of the insert relative to the origin of replication and the length of (CTG/CAG)n motif.51 A widely accepted model (Fig. 3) proposes that deletions occur if the hairpin is formed on the lagging strand template (so called orientation II). A single-stranded CTG region within the replication fork forms a thermodynamically stable hairpin, which is then bypassed by incoming DNA polymerase, that in turn produces deletions. Conversely, expansions arise as a consequence of secondary DNA structures being formed during lagging strand synthesis (orientation I). Slippage of the newly synthesized repeated DNA, the formation of CTG hairpins on the Okazaki fragments, realignment of the primer, and the idle synthesis of DNA polymerase result in large expansions.5052 Hairpins are believed to be favored on the lagging strand, but they could also occur on the leading strand. In studies employing the single-stranded bacteriophage replication model, (CTG/CAG)n, (CGG/CCG)n, and (GAA/TTC)n repeats underwent deletions during leading-strand synthesis.53 Interestingly, of all ten possible triplet repeats, CTG motifs are expanded a few times more frequently than the other ones.51 A variety of studies have confirmed a dramatic influence of replication on the genetic instability of TRS in bacteria and yeast.5456

Figure 3. Mechanism of the genetic instability of the TRS during replication.

Figure 3

Mechanism of the genetic instability of the TRS during replication. Formation of the hairpin structure on the newly synthesized Okazaki fragment, primer relocation and idling synthesis will result in large expansion while the bypass synthesis through (more...)

As expected, the instability of both deletion and expansion events was strongly affected by length of repeated sequences. Inserts of less than 20 CTG units, that are much less prone to form secondary structures, do not delete nor expand, while tracts consisting of approximately 50 units become unstable. This resembles the situation observed during the development of the disease in humans.

Transcription

All TRS related to human diseases are actively transcribed. Unwinding of the double-stranded DNA by moving RNA polymerase complex introduces locally high torsional stress, which leads to the formation of twin domains of differential DNA supercoiling, with the regions ahead and behind the polymerase having increased positive and negative supercoiling, respectively.57 The energy of negative superhelical turns may facilitate formation of unusual DNA structures from sequences with high propensities to undergo such a transition. Notably, it was shown that transcription could promote hairpin formation within repeating sequences in E. coli, and formation of such structures in TRS during transcription could lead to length changes of the repeat tract. Studies in E. coli have shown also that transcription has a large impact upon the genetic stability of (CTG/CAG)175.58,59 Multiple recultivations of strains harboring TRS containing plasmids led to significant reduction of the full-length, non-deleted repeats under conditions where transcription through the repeat was induced. Similarly, transcription was found to destabilize dinucleotide repeats in yeast.60 Transcription was also reported to be crucial in affecting the genetic instability of long CTG/CAG motifs by NER pathway in E. coli.59

A possible correlation between replication, orientation of the TRS, active transcription, structural properties of repeated DNA and the genetic instability of TRS is shown in Figure 4. The top strand of the duplex TRS on both sides of the figure serves as the transcribed strand, as well as the leading-strand template for DNA synthesis. The left side of Figure 4 represents orientation II (CTG strand serves as the lagging strand template), whereas the right side shows orientation I (CTG is within the Okazaki fragment). Transcription of the CAG strand leads to deletions, whereas transcription of the CTG strand elicits a much lower frequency of deletions. The model proposes that as the CAG strand is being transcribed, the complementary CTG strand while being single-stranded, folds back and forms a hairpin. On the other hand, the non-transcribed CAG strand in orientation I is less able to form stable hairpins. Additionally, in orientation I the CTG strand is not single-stranded and cannot form stable hairpins because it is “occupied” by the RNA polymerase complex. The model further envisages that while TRS is transcribed, it is also replicated. In this case, the CTG hairpin in orientation II will be bypassed by the DNA polymerase complex during lagging-strand synthesis, and this will lead to deletions. Conversely, deletions in orientation I will be found rarely since there is a lower propensity to form secondary structures on the lagging-strand template by the CAG tracts and thus, no bypass synthesis occurs.

Figure 4. Correlation between replication, orientation of the (CTG/CAG)n tract, transcription, structural properties of repeated DNA and the genetic instability of the TRS.

Figure 4

Correlation between replication, orientation of the (CTG/CAG)n tract, transcription, structural properties of repeated DNA and the genetic instability of the TRS. Independently of the TRS orientation the moving transcription complex causes the opening (more...)

DNA Repair

Methyl-Directed Mismatch Repair (MMR)

In all organisms genomic integrity is normally maintained by a variety of DNA repair pathways, including MMR and nucleotide excision repair (NER).61 Secondary DNA structures formed during DNA synthesis, especially within single-stranded regions containing repetitive tracts, may be hazardous for genome stability if not removed by repair activities. MMR pathway is a fundamental system involved in maintaining genomic integrity because in addition to correcting mismatched base pairs, it also repairs some nonclassical DNA structures such as small hairpins and unpaired regions within DNA. Upon inactivation of MMR increased heterogeneity is observed at simple repetitive DNA (e.g., mono- and dinucleotides) in bacteria, yeast and mammals. The associations of defective MMR and an elevated genetic instability at simple DNA repeats are particularly strong for hereditary nonpolyposis cancer.

The investigations of a role of methyl-directed mismatch repair in TRS instability were an important step in studying the molecular mechanisms leading to the accumulation of dynamic mutations among triplet repeats. Notably, studies performed in bacteria and yeast have identified that MMR had contrasting effects on the genetic stability of TRS.6264 Although instability of the TRS is linked to human disorders, functional similarities between the MMR in prokaryotes, lower eukaryotes and humans justified this study. For example, E. coli strains with defective MMR had a reduced occurrence of large deletions (more than 8 repeats) from plasmids harboring long CTG/CAG. By contrast, mutations in MMR proteins increased the frequency of small length changes (less than 8 repeats) in shorter CTG/CAG repeats in E. coli and S. cerevisiae.

To clarify these apparently conflicting results, Parniewski et al have used a variety of lengths of CTG/CAG tracts (ranging from 25 to 175 units) to determine the effects of MMR on repeat tract stability in E. coli.65 They showed that depending on the length of repeats the functional MMR proteins act to promote large deletions (usually more than 8 repeats) in CTG/CAG tracts, but significantly prevent length changes (both, expansions and deletions) of less than 8 repeats. Not only the length of the TRS influenced the incidence of deletions in CTG/CAG but also the instability was dependent on the purity of TRS (i.e., presence of interruptions) as well as the cell growth conditions. One plausible explanation of this distinctive behavior of the MMR proteins acting on the TRS is the propensity of the triplet repeats to undergo different structural transitions depending on length of the repeated motif. Since short TRS are more likely to form slipped structures as opposed to the long ones, which will rather tend to assemble into stable hairpins therefore, different local non-B-DNA structures may trigger particular cellular mechanisms. Considering this and results from other groups, we propose a model which links structural properties of the (CTG)n to the polymerase pausing and bypass synthesis within DNA tracts being repaired by the MMR (Fig. 5). Following the DNA slippage of the complementary strands in double-stranded TRS region, small loops are formed on both strands and therefore are recognized by functional MMR proteins. The repair process leads to the excision of large segments of non-methylated strand spanning a region containing loopouts and to the formation of single-stranded regions on the complementary strand. Short single-stranded TRS tracts (of less than approx. 100 units) are much less prone to form stable hairpins than the long ones and resynthesis of the complementary strand will result in neither deleted nor expanded TRS. In the absence of functional MMR proteins, the same tract will be subjected to SILC pathway and consequently small expansions and deletions will gradually accumulate within the repeated motif after subsequent generations of cells. Therefore, repair of small loops that could arise on relatively short CTG/CAG tracts would stabilize the TRS and lack of this repair function will have an opposite effect. Conversely, the same repair pathway acts differently on long tracts of the TRS. Following the formation of slipped-stranded structures, recognition of small loops and excision of one DNA strand by the MMR protein complex will result in long single-stranded stretches in the CTG region which will self-pair and form stable hairpins. If during resynthesis of a gap DNA polymerase bypasses the hairpin the “repaired” molecule would contain big deletions. However, when the CAG strand serves as a template for repair synthesis (inverse orientation of CTG/CAG tract) the nascent DNA would be able to produce stable hairpins, which would possibly cause DNA polymerase to stall. Further multiple polymerase slippages, the relocation of newly synthesized repeated DNA fragment and idling synthesis will result in large expansions of the TRS.

Figure 5. The effect of methyl-directed mismatch repair on the genetic stability of short and long CTG/CAG tracts in generating SILC and MLC (see text for details).

Figure 5

The effect of methyl-directed mismatch repair on the genetic stability of short and long CTG/CAG tracts in generating SILC and MLC (see text for details).

Our model presented above explains opposite results concerning the role of the MMR system in generating TRS instabilities in bacteria and yeast, obtained in different laboratories. However, how MMR affects the frequency of expansion events in humans remains unclear. Moreover, long CAG/CTG repeats from the gene associated with Huntington's disease in humans were shown to be less prone to expand in transgenic mice with defective MSH2 protein.66 Together, these in vivo observations suggest that mutations in MMR enzymes are not required for expansions of TRS in mammals, and the involvement of this repair system in TRS related diseases needs to be more extensively studied.

Nucleotide Excision Repair (NER)

Nucleotide excision repair is another major cellular defense system in both prokaryotes and eukaryotes. This pathway efficiently recognizes and repairs a vast majority of damages, including bulky DNA adducts and DNA cross-links that cause significant distortion of the helix, as well as less distortive lesions such as methylated bases. Also, the involvement of NER in the repair of DNA loops in vitro has been reported.67,68 In humans, defects in NER proteins cause at least three hereditary disorders, including Xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy.61

Since unusual DNA structures can form in some TRS in vivo and may therefore invoke destabilization of double-stranded helix, they are also likely to trigger the NER proteins and during the repair process enhance repeat tract instability. Studies in E. coli revealed that bacterial NER proteins influence the genetic stability of the TRS in a complex manner.69 First of all, the stability, as demonstrated by previous investigations was highly dependent on the length of the repeated tract and the orientation of TRS insert relative to the origin of replication. The instability was only observed for long CTG/CAG tracts (175 units) in orientation II, where the CTG strand served as a template for lagging strand synthesis. However, in long-term (multigenerational) growth of the wild-type strain and its isogenic uvrA or uvrB mutants, the rate of deletions in strain lacking functional UvrA protein was significantly higher as compared to strain that lacked only UvrB. In E. coli UvrA is required for damage recognition. The affinity of the UvrA protein to single-stranded DNA, specifically to bubbles and loops may be responsible for the recognition and binding to the CTG hairpins in their single-stranded loop region.67,70 Binding of the UvrA to unusual conformations may destabilize such structures, allowing the correct copying of the entire repeat. Others have demonstrated that absence of the single-stranded-DNA-binding protein (SSB) in vivo similarly led to an increased frequency of large deletions within the triplet repeats.71 Very high stability of long CTG/CAG tracts in strains lacking functional UvrB suggests that this protein may be involved in processing of unusual structures within repeats and allows deletions to occur. In some in vitro studies specific recognition and excision of bubbles within double-stranded DNA by the UvrBC endonucleolytic complex was demonstrated.72 An alternative scenario is that in the absence of the UvrA protein, the CTG hairpin may be also a substrate for the cellular endonucleolytic activities. Such nicked DNA may be degraded in vivo, which would similarly lead to deletions. The possible pathways of the CTG hairpins processing by NER are presented on Figure 6.

Figure 6. The possible pathways of the NER-generated genetic instability of long transcribed CTG/CAG tracts in orientation II.

Figure 6

The possible pathways of the NER-generated genetic instability of long transcribed CTG/CAG tracts in orientation II. The CTG hairpin formed on the lagging-strand template during TRS replication (shaded circle) may be removed by the UvrA dimer (left panel), (more...)

Interestingly, the genetic differences in the stability of long CTG/CAG tracts between uvrA and uvrB mutants were apparent only if the TRS were transcribed. Transcription through the TRS may additionally stabilize CTG hairpins by introducing negative supercoils behind RNA polymerase complex. It is important to note that the NER pathway is well suited to repair transcribed strands. Any kind of RNA polymerase pausing triggers transcription-repair coupling factor (TRCF). This protein attracts NER components to the transcribed region, providing prompt removal of DNA lesions. One might assume that formation of the hairpin structures on the template strand as well as on the nascent RNA may lead to RNA polymerase stalling. Napierala et al demonstrated that CUG repeats form extremely stable, length-dependent, self-complementary structures. This strongly supports the hypothesis that structural aberrations within the TRS are causative for their genetic instability.

Recombination

Recombination is a major source of the genetic instability of all organisms. This process allows the cell to change the order of its genes, to move the sequence from one place to another (translocations), change the orientation (inversions), multiply (duplications) or remove (deletions) from the genome. However, one must remember that it serves also very often to repair damaged DNA.

Several pathways of recombination are known of which the most frequent one is the homologous recombination dependent on RecA protein in case of bacteria or its eukaryotic equivalents (i.e., Rad51p family in yeast). This process occurs usually between very similar or identical DNA tracts located on two different DNA molecules and positioned in the same regions with respect to the entire molecule (allelic recombination) and basically serves to keep the genome stable. Sometimes however, it may involve the sequences dispersed among the same chromosome for example direct and inverted repeats or the sequences located on nonhomologous replicons (ectopic or homeologous recombination). Interactions between such sequences lead to the gross genome rearrangements mentioned above.

The stability of the repeating sequences such as micro- and minisatellites has been investigated for several years and recently recombination has been shown as the second (along with the replication) major mechanism responsible for the contractions as well as the expansions of such tracts. This progress has been made particularly due to the improvement of the in vivo genetic assays allowing precise investigations of several pathways of recombination in eukaryotic cells, especially in yeast.

Among different recombination pathways, gene conversion, i.e., nonreciprocal transfer of the genetic information from one DNA duplex to another leading to non-Mendelian segregation is suggested to be the major source of the repeating sequences instability. This process is sometimes associated with cross-over events and the proportion of the gene conversions that are accompanied by the crossing over seems to be much higher during meiosis than mitosis.73,74 Different groups have explained mechanisms of gene conversion occurring without as well as with gene crossing-over, although the common feature of all models was the initiation of the process by the double strand break (DSB) within one duplex of DNA. A complex model has been proposed by Szostak et al75(Fig.7, panel A). This model assumes that DSB formation followed by the exonucleolytic digestion of the 5' ends leads to the formation of large gaps with 3' overhangs (up to 1kb or even more) which can invade a homologous template in order to repair broken DNA.76 Both 3' ends serve as the priming site for DNA polymerase, which elongates them using undamaged duplex as the template. After formation of the Holliday junctions, these four-stranded, branched structures may migrate in both directions, spanning bigger regions and ultimately may be resolved by cutting in one of two orientations. If the noncrossover strands are cut in one Holliday junction and crossed strands are cut in another, this gives rise to the gene conversion associated with the crossing over. Alternatively, if both junctions are cut in the same orientation, resulting gene conversion will not be associated with crossover. Such a concept assumes that the ratio between both types of events should be equal. Although several experimental data support this model, some work has revealed that this ratio is strongly biased towards the noncrossover products, especially during mitosis.7779 One explanation could be that there is some preference during the resolution of the Holliday junctions which leads to the same way of cutting in both structures (i.e., the resolution requires isomerization which allows only crossed strands to be cleaved).80,81 Other alternatives assuming that there is no need for Holliday junction formation in order to perform gene conversion have been proposed based on the analysis of the recombination products in such different organisms as E. coli, yeast, Ustilago, Drosophila as well as in humans.78,79,8286This alternative model of DSB repair called Synthesis-Dependent Strand Annealing (SDSA) assumes that after strand invasion both 3' ends of the donor are extended by DNA polymerase while the donor DNA remains unchanged.74,87 Thus, in contrast to the commonly known semiconservative character of the DNA replication, here the strand synthesis becomes a conservative process as it occurs on two strands within one duplex. The newly synthesized strands unwind from their templates and reanneal back within the broken duplex (Fig. 7, panel B). Both unique as well as the repeated sequences may recombine via this pathway, although in the case when the repeated sequences are involved, expansions and deletions may occur. This is due to the fact that after elongation newly synthesized strands contain two or more repeats which during reannealing may pair in out of frame order (Fig. 7, panel C). One must mention that SDSA models, although they explain the bias towards noncrossover events, do not exclude the possibility of crossover occurrence. If both strands of the recipient molecule are used as a synthesis start point, then it may lead to the formation of the two Holliday junctions so the gene conversion may be followed by the crossover as in the classical view of Szostak's model.

Figure 7. Recombination pathways of Double-strand Break repair.

Figure 7

Recombination pathways of Double-strand Break repair. (Frame) Double-strand breaks initiate nearly all homologous recombination pathways and are the start point for the 5'Æ3' exonucleolytic digestion which leaves 3' ends of donor DNA duplex (shown (more...)

For several years the mechanism of general homologous recombination has been considered to play an important role also in the instability of the trinucleotide repeats. Although the crossover mechanism seemed to be rather unlikely as no exchange of the flanking sequences has been observed, other recombination events like gene conversion or unequal crossover were suggested to be responsible for the triplet repeats size alterations observed in humans, mainly in myotonic dystrophy and fragile X patients.8892 Triplet repeats, although belonging to the category of microsatellites, feature some distinct characteristics which make them specific substrates for the recombination machinery. It has been established that double strand breaks may occur very frequently within these sequences. One reason for this could be the fact that the replication fork moving across long TRS may frequently pause leading to the formation of the unfinished Okazaki fragments.40,41 Such regions may induce the formation of the double strand breaks. The process of DNA polymerase stalling facilitates also the formation of the secondary structures on such incompletely synthesized Okazaki fragments. On the other hand, such structures may also form on the template strand, as it remains single-stranded. Besides the important role of the replication-based instability, which happens during formation of alternative secondary conformations (polymerase slippage), these structures also may be recognized by the specific cellular endonucleases resulting in DSB formation and subsequent increased recombination rates.93,94

Many experimental assays have been developed recently in order to analyze specifically the destabilizing effect of the recombination machinery on TRS. In bacteria, a system involving two-plasmid model has been used where two otherwise nonhomologous vectors both carrying TRS of different length were introduced into the recA background.95,96 Analysis of the recombination products using restriction mapping and sequencing revealed that CTG/CAG tracts were better substrate for gene conversion mechanisms than CGG/CCG tracts. Only long CTG/CAG tracts (more than 30 repeats) were recombinogenic and yielded with the multiple-fold-expanded products. Moreover, the recombination-induced expansions were much more frequent than deletions (approximately 100:1 ratio) in strong opposition to the results obtained when the TRS instability was induced by the replication mechanisms (1:100 ratio). The observation obtained with this model has not yet been confirmed though, as the attempts of another group to get CTG/CAG expansions using the similar system showed, there was a replication but not recombination-dependent character of the expansions.97

Several systems have been also established in order to observe TRS instability in yeast. Fungi historically served as the model organism in the recombination study as they allow for easy analysis of the products formed in mitosis as well as in meiosis. Therefore they are ideally suited for investigations of TRS instability as it is now thought that this process takes place in humans during meiosis as well as in early postzygotic stages of mitotic cell growth.98 It has been shown that most mitotic and basically all meiotic recombination processes in yeast are induced by double-strand breaks although the proteins as well as the mechanism of recombination occurring during these events are different.74 Indeed, the instability of TRS was observed as the recombination outcome of both types of divisions, although CTG/CAG tracts were much more prone to give deletions as well as expansions during meiosis, while mostly deletions were observed after mitosis.99,100 This process was dependent on the activity of the topoisomerase II-like trans-esterase Spo11, which induces the formation of the DSB within triplet repeats during meiosis.101 Only long tracts were recombinogenic while the shorter ones (10 repeats) were much more stable.99,102 Analysis of the mitotic recombination events revealed that the bias towards expansions or deletions of the CAG/CTG repeats was dependent on the length of such regions. Shorter tracts (39 repeats) yielded contractions, and the longer ones (98 repeats) showed both deletions as well as expansions.103 On the other hand, in the case of CCG/CGG repeats, no difference in stability was observed between mitotic and meiotic events regardless on the length of the repeated tracts.104

The study of the recombination-induced TRS instabilities are also currently ongoing using mammalian models including human cells. Several yeast protein homologues involved in the general homologous recombination between TRS as well as other sequences have been identified in higher eukaryotes. Some of them seem to play an even more essential role than in yeast since their absence may give very severe phenotypes in vertebrates. Among them, homologues of Rad51p (which deletion is lethal for vertebrates but not for yeast) and its related proteins Rad55p and Rad 57p, as well as Dmc1, Rad54p, Rad52p, Mre11, Rad50, Xrs11 have been described. Also the homologues of the yeast proteins engaged specifically in the meiotic recombination like Spo11, Msh4p, Msh5p have been identified (for a review see refs. 74, 105). Analysis of the mutants lacking such proteins indicates that in the higher eukaryotes they play a similar role (although in some cases a slightly distinct role) and thus one may predict their involvement in the instability of the triplet-repeat sequences in humans. Nevertheless, more specific data on the recombination-induced TRS expansions based on the analysis of vertebrate models, especially human cells, is highly desired.

It is also worth mentioning that instability of the repeated sequences (possibly including TRS) may result from the activity of recombination pathways not described in this section. For example, some experiments have demonstrated that the recombination between direct repeats located in close proximity can occur efficiently in a RecA-independent manner in E. coli cells. The frequency of this process was dependent on the length of the direct repeats as well as the distance between them. Moreover, these factors had the impact on the outcome of the process as the short neighboring repeats yielded monomeric products, whereas the increase of length as well as the distance between the repeats gave rise to the two different kinds of dimeric products. The model in which the misalignment of the direct repeats during DNA replication forms highly recombinogenic substrate, which can be then processed by different RecA-independent pathways, may explain this observation.106

Finally, the involvement of the illegitimate recombination induced by side-products of the activity of some enzymes (i.e., topoisomerases) should be taken into consideration.

Summary

To date 14 neurodegenerative disorders including myotonic dystrophy, Huntington's disease, Kennedy's disease, fragile X syndrome, spinocerebellar ataxias or Friedreich's ataxia have been linked to the expanding trinucleotide sequences. Although phenotypic features vary among these debilitating diseases, the structural abnormalities of the triplet repeat containing DNA sequences is the primary cause for all of these disorders. Expansions of the CAG repeat within coding regions of miscellaneous genes result in the synthesis of aberrant proteins containing enormously long polyglutamine stretches. Such proteins acquire toxic functions and/or may direct cells into the apoptotic cycle. On the other hand, massive expansions of various triplet repeats (i.e., CTG/CAG, CGG/CCG/, GAA/TTC) inside the noncoding regions lead to the silencing of transcription and therefore affect expression of the adjacent genes. The repetitive character of TRS allows stretches of such tracts to form slipped-stranded structures, self-complementary hairpins, triplexes or more complex configurations called “sticky DNA”, which are not equally processed by some cellular mechanisms, as compared to random DNA.

It is likely that the instability of the short TRS (below the threshold level) occurs due to the SILC pathway, which is driven by the DNA slippage. Accumulation of the short expansions leads to the disease premutation state where the MLC pathway becomes predominant. Independent of which mechanism is involved in the MLC pathway (replication, transcription, repair or recombination) the process of complementary strand synthesis is crucial for the TRS instability. Generally, dependent on the location of the tract which has higher potential to form secondary DNA structure, further processing of such tract may result in expansions (secondary structure formed at the newly synthesized strand) or deletions (structure present on the template strand).

Analyses of molecular mechanisms of the TRS genetic instability using bacteria, yeast, cell lines and transgenic animals as models allowed the scientists to better understand the role of some major cellular processes in the development of neurodegenerative disorders in humans. However, it is necessary to remember that most of these investigations were focused on the involvement of each particular process separately. Much less of this work though was dedicated to the search for the interactions between such cellular systems that in effect could result in different rate of TRS expansions. Thus, more intensive studies are necessary in order to fully understand the phenomenon of the dynamic mutations leading to the human hereditary neurodegenerative diseases.

Acknowledgments

Authors were partially supported by the State Committee for Scientific Research (KBN grant 6P04A 01617).

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