Nucleotide excision repair affects the stability of long transcribed (CTG•CAG) tracts in an orientation-dependent manner in Escherichia coli (original) (raw)

Journal Article

,

Search for other works by this author on:

,

Search for other works by this author on:

,

1

Center for Microbiology and Virology, Polish Academy of Sciences

,

106 Lodowa Street, 93-232 Lodz, Poland

Search for other works by this author on:

*To whom correspondence should be addressed. Tel: +1 713 677 7651; Fax:

+1 713 677 7689

; Email: rwells@ibt.tamu.edu

Search for other works by this author on:

Accepted:

15 November 1998

Published:

01 January 1999

• PDF
• Split View
• • Article contents
  • Figures & tables
  • Video
  • Audio
  • Supplementary Data
• Cite

Cite

Pawel Parniewski, Albino Bacolla, Adam Jaworski, Robert D. Wells, Nucleotide excision repair affects the stability of long transcribed (CTG•CAG) tracts in an orientation-dependent manner in Escherichia coli, Nucleic Acids Research, Volume 27, Issue 2, 1 January 1999, Pages 616–623, https://doi.org/10.1093/nar/27.2.616
Close

Navbar Search Filter Mobile Enter search term Search

Abstract

The influence of nucleotide excision repair (NER), the principal in vivo repair system for DNA damages, was investigated in Escherichia coli with uvrA, uvrB and uvrAuvrB mutants with the triplet repeat sequences (TRS) involved in myotonic dystrophy, the fragile X syndrome and Friedreich's ataxia. (CTG•CAG)175 was more stable when the (CTG) strand was transcribed than when the (CAG) strand was transcribed in the alternate orientation. A lack of the UvrA protein dramatically increases the instability of this TRS in vivo as compared with the stability of the same sequence in uvrB mutant, which produces an intact UvrA protein. We propose that transcription transiently dissociates the triplet repeat complementary strands enabling the nontranscribed strand to fold into a hairpin conformation which is then sufficiently stable that replication bypasses the hairpin to give large deletions. If the TRS was not transcribed, fewer deletions were observed. Alternatively, in the uvrA− mutant, the hairpins existing on the lagging strand will suffer bypass DNA synthesis to generate deleted molecules. Hence, NER, functionally similar in both prokaryotes and eukaryotes, is an important factor in the genetic instabilities of long transcribed TRS implicated in human hereditary neurological diseases.

Introduction

Neurogenetic diseases including myotonic dystrophy (DM), fragile X syndrome, Huntington's disease, spinobulbar muscular atrophy, spinocerebellar ataxia type 1 and Friedreich's ataxia result from expanded triplet repeat sequences (TRS) [(CTG•CAG), (CGG•CCG) or (GAA•TTC)] within their genes (1,2). The lengths of the TRS influence the age of onset and the increased severity of the neurological diseases through family pedigrees (clinically referred to as anticipation). Long tracts of TRS are unstable and show repeat size polymorphisms in successive generations and in different tissues. The expansion events involved in the etiology of hereditary neurological diseases fall into two categories (Type 1 and Type 2) (2). For relatively small TRS expansions (Type 1 diseases) involving expanded glutamine tracts in the target proteins, small slipped-register expansions and deletions (SSED) may be responsible (3). However, for the massive expansions involved in the Type 2 diseases such as DM and fragile X, a different mechanism involving replication errors (4–10) or recombination (11–16) is likely. In addition to observations in humans, TRS instabilities have been demonstrated in Escherichia coli (5,6,17–23), yeast (4,11,24), transgenic mice (25) and cultured cells from patients (26,27).

The molecular mechanism responsible for the genetic instability of TRS is influenced by many factors (1,8). The availability of genetically and biochemically defined systems in E.coli enabled detailed investigations of the TRS instabilities. Deletions and expansions occur in E.coli (5,6) and TRS instability is influenced by the direction of replication through the TRS (5,6,18,19). Also, a destabilizing effect of active transcription into long (CTG•CAG) tracts was demonstrated (18). Different growth conditions, especially entering a stationary phase by E.coli harboring (CTG•CAG) tracts (19), causes increased deletions of the TRS. Functional single-stranded DNA-binding protein (SSB) was an important cellular factor in maintaining the genetic stability of triplet repeats in E.coli (22).

Numerous prior investigations have shown the occurrence of minisatelite instabilities caused by inactivation of methyl directed mismatch repair (MMR) or nucleotide excision repair (NER) (28–39). Much less is known, however, about the involvement of the repair systems in TRS instability (3,17,24,40). Long (CTG•CAG) repeats are stabilized in plasmids in E.coli with mutations in the MMR genes (mutS, mutL or mutH). Mutations in these genes prevent large (approximate multiples of 40 repeats) TRS deletions. However, in the same genetic background, SSED occur very frequently (3). Similar data also were reported for pms1 and msh2 yeast mutants (24).

NER is the major cellular defense system in both prokaryotes and eukaryotes (41). Defects in excision repair cause three human diseases (42): xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy. The substrate specificity of the E.coli Uvr(A)BC endonuclease is very wide and includes bulky DNA adducts and DNA cross-links that cause significant distortion of the DNA helix as well as less distortive lesions such as methylated bases (43,44). The involvement of NER in the repair of DNA loops has also been reported (37,45).

Herein, we demonstrate that the deficiency of some NER functions dramatically affects the stability of long (CTG•CAG) inserts in plasmids in E.coli. This stability is strongly dependent on the orientation of the (CTG•CAG) tract, relative to the ColE1 origin of replication, and on active transcription into the TRS.

Materials and Methods

Plasmids

All plasmids used in these experiments containing repeating (CTG•CAG)n or (CGG•CCG)n or (GAA•TTC)n inserts are shown in Table 1.

Table 1

Plasmids used in this study

pRW3247 and pRW3248 are derivatives of pUC19 _Not_I and were described previously (5,6,17–19). The plasmids contain either (CAG)175 (pRW3247, orientation II) or (CTG)175 (pRW3248, orientation I) as the leading strand template for replication. This sequence is not homogeneous but contains two G to A interruptions at repeats 28 and 69. Plasmids pRW3268 and pRW3269 contain the same insert cloned in both orientations but the vector lacks the _Pvu_II fragment containing the LacZ′ promoter region such that the (CTG•CAG) insert is not transcribed (5,17,18). pRW4011 is a derivative of pUC18 and contains an uninterrupted (CAG•CTG)50 cloned in orientation II. pRW3032 is a pUC19 _Not_I-based plasmid containing the (CGG•CCG)24 insert cloned into the _Bam_HI site, where the CCG is in the leading strand template (20). pRW4006 is a pUC18 _Not_I-based plasmid containing the same insert cloned in orientation II, where the CGG is in the leading strand template. pRW3804 is a derivative of pUC18 _Not_I (46) containing the (GAA•TTC)70 fragment (47) inserted into the _Bam_HI site of the vector. The plasmid was obtained by the in vivo expansion technique (47); the GAA strand is in the leading strand template. pRW3803 is a derivative of pUC19 _Not_I and the (TTC)65 is in the leading strand template. These two plasmids were constructed and characterized by K. Ohshima (47).

Bacterial strains

To study the influence of the NER system on the instability of the TRS, the following E.coli strains were used: AB1157 [thr-1, ara-14, leuB6, D(gpt-proA)62, lacY1, tsx-33, qsr′ -, glnV44(AS), galK2, l, arc- hisG4(Oc), rfbD1, mgl-51, rpsL31(sm R _), kdgK51, xylA5, mtl1, argE3(Oc), thi-1_] as a parent (wild-type) of the NER deficient strains; E.coli AB1886 is isogenic with AB1157 except for uvrA6; E.coli AB1885 is isogenic with AB1157 except uvrB5; E.coli AB2421 is isogenic with AB1157 except uvrA6uvrB45. Strains AB1157, AB1886 and AB1885 were obtained from the E.coli Genetic Stock Center. Escherichia coli AB2421 strain was a gift of Dr E. Tang (University of Texas, M. D. Anderson Cancer Center, Science Park, Research Division, Smithville, TX).

Conditions of bacterial growth

The plasmids containing various TRS were transformed into the appropriate E.coli strain and grown for a number of generations, as described (19). Briefly, E.coli cells were transformed with the plasmids and an aliquot of this mixture was inoculated into 10 ml of LB containing ampicillin at 100 µg/ml. Incubations of the liquid cultures were continued overnight at 37°C at a shaking rate of 250 r.p.m. The bacteria then were subcultured into fresh liquid media with a dilution factor of 107. The cells from each culture were harvested and plasmids were isolated.

Analysis of DNA

The undeleted plasmids were isolated by elution of the full-length supercoiled DNA from the appropriate location on the agarose gel. Transformation of E.coli cells with the DNA was performed using electroporation (Electroporator 1000, Stratagene). DNA preparations, agarose and polyacrylamide gel electrophoreses were performed according to standard laboratory protocols (48). Plasmids were prepared by alkaline lysis of 10 ml cultures using the standard alkaline lysis miniprep procedure. Restriction digests were performed following the manufacturer's instructions. All plasmids except for pRW3268 and pRW3269 were digested with _Eco_RI and _Hin_dIII. pRW3268 was cleaved with _Eco_RI and _Nde_I and pRW3269 was cleaved with _Eco_RI and _Afl_III. The restriction analyses were performed by electrophoresis through 7 or 6% polyacrylamide gels in TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0) buffer. The gels were stained with ethidium bromide and photographed. The negatives of the gels were scanned (300S, Molecular Dynamics) and the amount of the TRS containing fragment at full-length was quantified. For some experiments the restriction digests were radiolabeled using E.coli DNA polymerase I Klenow fragment incubated with [α-32P]dATP, subjected to PAGE and radiolabeled fragments were observed by autoradiography or with a PhosphorImager (400S, Molecular Dynamics). Quantitation of these gels was performed directly on the phosphorimage. The percent of full-length of (CTG•CAG)175 was determined relative to all smaller fragments of DNA on the gel.

Results

Effect of orientation of (CTG•CAG)175 on its stability in NER proficient and deficient cells: orientation II

The effect of NER on the instability of the (CTG•CAG)175 insert cloned in both orientation I and orientation II in plasmids (Table 1) was determined. The orientation of these TRS with respect to the direction of replication within these plasmids was as follows: plasmids containing the CTG sequence as the leading-strand template are designated orientation I; plasmids containing the CAG sequence as the leading-strand template are designated orientation II (5). Several prior studies (5,17–19) revealed that the TRS in orientation I is substantially more stable due to the replication behavior on CTG hairpin loops which are relatively more thermodynamically stable than CAG hairpin loops (49–53). Genetically defined E.coli strains were used (see Materials and Methods). The strains containing mutations in the uvrA or uvrB or uvrA and uvrB (double mutant) genes as well as the isogenic wild-type E.coli were transformed with pRW3247 (TRS in orientation II) or pRW3248 (TRS in orientation I). The cells were grown overnight and then subcultured into fresh LB medium with a dilution factor of 107. After three recultivations of the cells, the DNA from each culture was isolated and plasmids were cleaved with _Eco_RI and _Hin_dIII to release the insert containing the TRS. Restriction digests were analyzed by 7% PAGE (Fig. 1). The quantitation of deletions found for pRW3247 and pRW3248 in the wild-type, uvrA and uvrB strains is shown in Figure 2A.

Figure 1

Effect of orientation on instability of transcribed (CTG•CAG)175 tracts in plasmids in wild-type E.coli and NER mutant strains. The E.coli strains were transformed with purified non-deleted monomer of pRW3247 (TRS in orientation II) or pRW3248 (TRS in orientation I) and analyzed as described in the Materials and Methods. The numbers above the samples indicate the number of subcultures. An arrow points to the band corresponding to the full-length, non-deleted (CTG•CAG)175. The 1 kb ladder was purchased from Gibco BRL.

A gradual disappearance of the restriction fragment corresponding to the full-length, non-deleted (CTG•CAG)175 was observed as analyzed for wild-type cells harboring pRW3247 (TRS in orientation II; Figs 1 and 2, left panel). These data are consistent with the previously reported behavior of the same plasmid as investigated in HB101 and DH5a E.coli strains (5,17–19). However, if this plasmid was propagated in cells having a mutation in the uvrA gene (E.coli 1886), a dramatic increase in the instability of the (CTG•CAG)175 insert was observed. The amount of the full-length non-deleted TRS calculated after the first recultivation of the uvrA cells harboring pRW3247 was significantly reduced as compared to the amount of non-deleted TRS calculated for the wild-type cells (52 and 76%, respectively). After the second recultivation of the uvrA mutant harboring pRW3247, the full-length TRS containing fragment was almost completely deleted (1% of non-deleted insert), whereas the pRW3247 isolated from the wild-type cells still contained a significant amount of the full-length (CTG•CAG)175 (24% of non-deleted insert) (Fig. 2A).

The opposite behavior was observed if pRW3247 was grown and subcultured in uvrB cells (E.coli 1885). The stability of the (CTG•CAG)175 insert in this strain was strikingly high and, even after three recultivations of the cells, 45% of the plasmids contained the full-length TRS. After the same number of recultivations of the wild-type and uvrA cells harboring pRW3247, the stability of the TRS in these strains was very low (9 and 0% of full-length TRS fragment, respectively). The stability of the (CTG•CAG)175 in the uvrAuvrB double mutant after the first recultivation of the cells harboring pRW3247 was comparable to that observed in the wild-type strain (77 and 76%, respectively) (Fig. 1A) but the amount of the non-deleted TRS measured after the second growth was higher (48% for the uvrAuvrB strain and 24% for the wild-type). However, after three recultivations of the cells, the amount of the full-length (CTG-•CAG)175 in both the wild-type and double mutant strains was very low (9 and 6%, respectively) (Fig. 1A).

To determine whether the UV irradiation of the DNA (during the isolation of the full-length supercoiled DNA) influenced the genetic instability of the (CTG•CAG)175 in plasmids grown in NER proficient and deficient strains, we conducted similar experiments using DNAs that were not exposed to UV (results not shown). No substantial differences were observed between these experiments and the data presented above.

NER in E.coli requires both UvrA and UvrB proteins to be functional (41). Since E.coli 1886 (uvrA), E.coli 1885 (uvrB) and E.coli AB2421 (uvrAuvrB) mutants are NER deficient (54), our data suggest that different mechanisms affect the stability of the (CTG•CAG)175 in NER proficient and NER deficient cells. As shown in Figure 1, the stability of this TRS after the first and second recultivations was higher in the double mutant (both UvrA and UvrB proteins not present) as compared to the wild-type strain. However, the difference in the stability of the TRS between uvrA and uvrB strains, that both are NER deficient, was much more pronounced (Figs 1 and 2). Thus, it seems that the UvrA protein stabilizes the TRS containing insert whereas the UvrB protein, which is a DNA endonuclease, contributes to the instability of the long (CTG•CAG) tract.

Figure 2

Instability of (A) transcribed and (B) untranscribed (CTG•CAG)175 tracts in plasmids. The plasmids designated were transformed into the wild-type and NER mutants and the cells were grown for the number of recultivations indicated. Restriction digests were analyzed by PAGE as described for Figure 1 and the amount of non-deleted TRS was calculated. (A) Quantitation of deletions in pRW3247 and pRW3248 (TRS transcribed). Analyses were performed on digested samples fractionated through 7% PAGE in TAE (as shown in Fig. 1). Each point on the graph represents an average value from three experiments. (B) Quantitation of deletions in pRW3269 and pRW3268 (TRS not transcribed). Quantitation was performed on the digested samples, radiolabeled with [α-32P]dATP and separated by 7% PAGE. The reproducibility of the measurement of the amount of the full-length (CTG•CAG)175 was estimated to be ±2%. Open symbols, plasmids containing (CTG•CAG)175 cloned in orientation I; filled symbols, plasmids containing (CTG•CAG)175 cloned in orientation II; circles, wild-type E.coli; diamonds, uvrA mutant; triangles, uvrB mutant.

Effect of orientation of (CTG•CAG)175 on its stability in NER proficient and deficient cells: orientation I

Similar investigations were conducted on the same DM sequence in pRW3248 which contains the (CTG•CAG)175 insert cloned in orientation I (Table 1). The NER proficient (wild-type) and deficient (uvrA or uvrB or uvrAuvrB) cells were transformed with pRW3248 and grown for three consecutive subcultures as described. DNA from each culture was isolated and plasmids were cleaved with _Eco_RI and _Hin_dIII to release the insert containing the TRS. Restriction digests were analyzed by 7% PAGE (Fig. 1, right panel). The quantitation of deletion products in pRW3248 is shown in Figure 2A.

Restriction analyses of pRW3248 propagated in NER proficient and deficient strains clearly show that the difference in the stability of the (CTG•CAG)175 between the wild-type and NER− strains is limited to the TRS in orientation II, since no substantial amount of deletions were observed in all E.coli strains under investigation harboring pRW3248 (TRS in orientation I) (Fig. 1, right panel and Fig. 2A).

Effect of transcription on the instability of the (CTG•CAG)175

We showed previously that active transcription into long (CTG•CAG) tracts markedly increased the frequency of deletions of (CTG•CAG)175 from plasmids (18). Instability occurred when E.coli cells harboring pRW3247 were grown and subcultured through a number of generations. It is also known that transcription recruits NER to damaged regions of DNA (55–57). Thus, the following experiments were undertaken to evaluate the role of transcription into the TRS as a factor influencing the stability of the (CTG•CAG)175 in NER proficient and deficient E.coli strains.

Two pairs of plasmids containing the (CTG•CAG)175 insert were used: pRW3247 (TRS in orientation II, transcribed) and pRW3248 (TRS in orientation I, transcribed) that contain the (CTG•CAG)175 insert cloned in the polylinker of pUC19 _Not_I that contain intact promoter for LacZ′; pRW3268 and pRW3269 contain the same insert (cloned in orientation II and orientation I, respectively) but not located inside a transcription unit (Table 1). The NER proficient (wild-type) and deficient (uvrA or uvrB) cells were transformed with pRW3268 (TRS in orientation II) or pRW3269 (TRS in orientation I) and grown for five consecutive subcultures as described. DNA from each culture was isolated and plasmids were cleaved with _Eco_RI and _Nde_I (for pRW3268) or with _Eco_RI and _Afl_III (for pRW3269) to release the insert containing the TRS. Restriction digests were radiolabeled (see Materials and Methods), separated by 6% PAGE and analyzed with a PhosphorImager (data not shown). The amount of full-length, non-deleted (CTG•CAG)175 was plotted as a function of the number of subcultures (Fig. 2B). No substantial instability of the TRS was observed after five recultivations of the wild-type and NER− cells harboring pRW3269 (TRS in orientation I). After the same number of generations, the instability of the (CTG•CAG)175 insert in the NER proficient and deficient cells harboring pRW3268 (TRS in orientation II) was clearly detectable. These data show similar orientation-dependent instabilities of the two plasmids as reported for DH5a and HB101 E.coli strains (5,17–19).

Table 2

Transcription- and orientation-dependent instability of (CTG•CAG)175 tract in wild-type and NER mutants

Similar analyses were conducted for the wild-type and NER deficient strains harboring pRW3247 and pRW3248 (TRS transcribed). Figure 2A shows the percentage of full-length (CTG•CAG)175 calculated from experiments described earlier and presented in Figure 1. A comparison of the behavior of pRW3247 and pRW3248 (TRS transcribed) (Fig. 2A) and pRW3269 and pRW3268 (TRS not transcribed) (Fig. 2B) clearly shows that NER proteins in E.coli affect the stability of the (CTG•CAG)175 insert when the (CTG) strand is the lagging-strand template for replication but only if the TRS region is transcribed. The effect of orientation of the (CTG•CAG) tract and transcription going through the TRS after five recultivations of the strains under investigation is summarized in Table 2. These data clearly show the effect of the orientation on the stability of the TRS (pRW3268 versus pRW3269) and the influence of active transcription into the (CTG•CAG)175 in orientation II (pRW3247) in NER proficient and deficient E.coli strains. The explanation for the involvement of transcription as well as NER proteins in the stability of long (CTG•CAG) tracts in plasmids is shown in Figures 3 and 4 and will be discussed below (Discussion).

Instability of other lengths and types of TRS

Thus far, we have shown that the stability of a long transcribed (CTG•CAG) tract is influenced by mutations in NER genes in E.coli. The absence of the uvrA gene product greatly enhances the instability of the TRS whereas lack of the functional UvrB protein causes substantial stabilization of (CTG•CAG)175 in orientation II.

Figure 3

Model for the involvement of transcription and TRS orientation in the instability of the (CTG•CAG)n. Local positive and negative superhelical domains (indicated by pluses and minuses) caused by transcription (64,65) through the (CTG•CAG)n facilitate an opening of the duplex TRS. The single-stranded (CTG) region formed for orientation II (left side) is not complexed by RNA polymerase, as in the case of orientation I, and therefore this region is able to form the (CTG) hairpin. Since the DNA polymerase complex may bypass this hairpin during lagging-strand synthesis, this would lead to deletions of the (CTG•CAG)n tract in orientation II but not in orientation I (right side).

Additional experiments were performed to determine whether the stability of shorter (CTG•CAG) tracts as well as other repeated sequences, related to human neurodegenerative diseases, are susceptible to the mutations in NER in E.coli. To evaluate the effect of length of the (CTG•CAG) tract on its stability in NER proficient and deficient strains, the deletion properties of (CAG•CTG)50 in orientation II were investigated. Wild-type as well as uvrA or uvrB E.coli strains were transformed with the full-length non-deleted monomer of pRW4011 (Table 1) and grown for the indicated number of recultivations. The DNA from each culture was isolated and plasmids were cleaved with _Eco_RI and _Hin_dIII to release the TRS containing fragment. Restriction digests were analyzed by 7% PAGE (data not shown). The quantitation of the deletions (described in Materials and Methods) in pRW4011 for the wild-type, uvrA and uvrB E.coli strains is shown in Table 3. The extent of instability of (CAG•CTG)50 was essentially the same among all strains investigated. Also, studies performed on a plasmid containing (CTG•CAG)50 in orientation I (pRW4015) (not shown) showed a higher stability of the TRS and no differences between NER proficient and deficient strains were observed. These data demonstrate that for (CTG•CAG) tracts, besides the effect of the orientation and transcription into the TRS, a certain length of the (CTG•CAG) (>50 units) is required as an additional factor that triggers NER in E.coli.

Figure 4

Model for the involvement of the UvrA protein in the stabilization of (CTG•CAG)175_in vivo_. The lower part of the figure shows events occurring on the shaded region of the top replicating molecule. The (CTG) hairpin formed during replication of the lagging strand in orientation II is recognized by the UvrA protein (left side). The interaction of the dimeric UvrA (69) with the (CTG) hairpin causes the removal of the DNA secondary structure and thus obviates bypass synthesis through the (CTG) hairpin. This leads to substantial stabilization of the TRS and gives rise to undeleted DNA. However, for _uvrA_− cells (right side), bypass synthesis through the TRS containing the CTG hairpin (no UvrA protein) leads to deletions.

Similar investigations conducted on the fragile X sequence (CCG•CGG) cloned in both orientation II and orientation I in plasmids pRW3032 and pRW4006, respectively, showed no effect of the genetic background of the E.coli strains (Table 3).

The same behavior was observed for pRW3803 and pRW3804 (Table 1) harboring the (GAA•TTC) Friedreich's ataxia sequence cloned in both orientations. The stability of the (GAA•TTC) tracts showed the orientation dependent effect as described (47) but the mutations in uvrA and uvrB genes had no influence on the amount of deletions in these plasmids (Table 3).

Thus, our results for the (CGG•CCG) and (GAA•TTC) sequences cloned in plasmids show that these TRS may be too short and/or that some different structural properties of (CGG•CCG) and (GAA•TTC) (21,58,59) tracts do not induce the NER in E.coli. Other prior investigations have revealed the structural uniqueness of (CTG•CAG) relative to the other TRS (21,60).

Table 3

Instability of other lengths and types of TRS contained on plasmids grown in wild-type E.coli and NER mutants

Discussion

NER (41) has a substantial impact on the genetic stability of long (CTG•CAG) inserts cloned in plasmids in E.coli. NER is the major cellular defense system against a variety of DNA damages in both prokaryotes and eukaryotes (61,62). To investigate the potential influence of this system on the stability of the TRS in vivo, we employed E.coli NER mutants. In the wild-type AB1157 strain, the (CTG•CAG) tract cloned in orientation II, relative to the ColE1 origin of replication, is more unstable compared to orientation I, and its genetic instability is strongly dependent on active transcription through the TRS sequence. These results confirm previous observations obtained with the HB101 strain, and agree with our hypothesis (18) that this instability is caused by a collision of the replication apparatus with the transcription machinery, rather than by host-specific factors.

Figure 3 presents a model that shows how transcription and orientation of a (CTG•CAG) tract may affect its genetic stability. The top strand of the duplex TRS on both sides of the figure is the transcribed strand, as well as the leading strand template for DNA synthesis. The left side of Figure 3 represents orientation II, whereas the right side shows orientation I. Transcription of the (CAG) strand (orientation II) leads to deletions, whereas transcription of the (CTG) strand (orientation I) elicits a much lower frequency of deletions. The model proposes that as the (CAG) strand is being transcribed, the complementary (CTG) strand 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; these differences in thermodynamic stability are based on studies conducted on single-stranded oligonucleotides (63). The model further envisages that while the 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 (instability). Conversely, in orientation I, fewer deletions will be found since fewer secondary structures are formed on the lagging-strand (CAG) template and thus no bypass synthesis occurs.

Therefore, the crucial component of this model is the selective formation of hairpins on the non-transcribed (CTG) strand. Transcription is accompanied by an accumulation of positive supercoils in front of the RNA polymerase complex and negative supercoils behind it (64,65). It is also known that negative supercoiling unwinds the helical duplex DNA and promotes strand separation (66). Measurements of the elastic constants of (CTG•CAG)n and (CGG•CCG)n and calculations of their free energy of supercoiling (58,67,68) revealed their flexibility and their writhed structure, compared to random B-DNA. Hence, these TRS were proposed to act as a ‘sink’ for the accumulation of superhelical density (58,67). In addition to this superhelical tension (free energy of supercoiling), in orientation II the (CTG) strand is not complexed by RNA polymerase, as in the case of orientation I, but rather is transiently single-stranded. We suggest that this ‘transient single-strandedness’ of the (CTG) strand in orientation II enables the nucleation of a folded-back (CTG) region into a hairpin.

The results obtained for the NER deficient E.coli strains show that the absence of certain repair functions dramatically influences the stability of the long transcribed (CTG•CAG) tracts in plasmids. This was manifested by the higher stability of the TRS in the uvrB mutant (UvrA protein present) than observed in the uvrA strain. Figure 4 presents a model for the involvement of the UvrA protein in the stabilization of the long (CTG•CAG) sequence in vivo. The top part of the figure represents a replication fork for the TRS region in a plasmid containing the (CTG•CAG)175 in orientation II. The (CTG) hairpin formed during the lagging-strand synthesis (Fig. 3) is shown inside the shaded circle (which is the focus of the remainder of the figure). The left side of Figure 4 shows how the UvrA protein may contribute to the stabilization of the TRS. This part of the model is based on the data obtained from the analyses of the (CTG•CAG)175 containing plasmid as grown and analyzed in the uvrB mutant. This strain is NER deficient but is able to produce the functional UvrA protein. The model predicts that the UvrA protein specifically recognizes and binds the (CTG) hairpin formed during the lagging-strand synthesis (Fig. 4, left side). This binding of the dimeric UvrA (69) to the (CTG) hairpin causes the destabilization of the hairpin. The resulting molecule contains a single-stranded gap of the size of the previous hairpin. This restores the correct template for complementary strand synthesis. The gap is then filled by DNA polymerase, followed by the ligation at the nicks, and the resulting molecule contains the full-length, non-deleted (CTG•CAG) tract.

On the other hand, in the absence of the UvrA protein (uvrA E.coli strain) (Fig. 4, right side), the (CTG) hairpin will not be removed from the DNA template. Thus, bypass DNA synthesis through the TRS may occur, which would cause deletions. The affinity of the UvrA protein to single-stranded DNA (70), specifically to bubbles and loops (45), may be responsible for the recognition and binding to the (CTG) hairpins in their single-stranded loop region. Also, the increased instability of (CTG•CAG) inserts was shown in vivo in E.coli having a mutation in the ssb gene (22). The absence of the SSB in vivo led to an increased frequency of large deletions within the triplet repeats. Another possible scenario is that in the absence of the UvrA protein, the (CTG) hairpin is a substrate for the DNA nicking activities (71). Such nicked DNA may be degraded in vivo which would also lead to deletions. The greater stability of long (CTG•CAG) tracts in the uvrB mutant than observed in wild-type cells, as well as the difference in the stability of the TRS between wild-type cells and the uvrAuvrB double mutant, also suggests that the UvrB protein itself may possess some nicking activity toward the CTG hairpin loops.

We propose that the (CTG) hairpins share, at least to some extent, similar properties with a variety of the DNA lesions that are recognized and repaired by the NER system. The substrate specificity of the Uvr(A)BC endonuclease is very wide (41). The important factor involved in the recognition of lesions by UvrA is a distortion of the DNA structure. Cruciform DNA and three-way junction structures, that are basically similar to the hairpins formed by the (CTG) tracts, introduce a significant amount of bending into DNA (72,73). Recently, the triplet repeats were shown to be inherently flexible and writhed (58,67,68,74). Also, (CTG•CAG) tracts can form slipped structures (3,75) and under some circumstances appear as bent/kinked DNA molecules (75). Additionally, the unorthodox DNA conformations of the triplet repeats were shown to be responsible for the DNA polymerase pausing on those sequences in vitro (76,77). It is possible that such pausing in vivo causes the arrest of replication, the induction of the SOS response and, as a consequence, de-repression of NER protein synthesis. Also, it was shown that repair activity in HeLa extracts was induced by a triple helix, and the authors suggested that other non-duplex DNA structures might trigger repair systems (78).

Our results and the model (Fig. 4) are supported by recent discoveries demonstrating the involvement of the UvrA protein in blocking the DNA repair-dependent _trans_-lesion synthesis pathway operating in E.coli (79,80). The binding of the UvrA protein to DNA containing a UV lesion in a single-stranded DNA prevented the bypass synthesis. In the absence of the UvrA protein the _trans_-lesion synthesis was not inhibited and this process is believed to be responsible for SOS mutagenesis in E.coli (41). In our case, however, binding of the UvrA protein to the (CTG) hairpin may be sufficient to remove the ‘lesion’. Such binding destabilizes the (CTG) hairpin, restores a ‘non-mutated’ template for replication and therefore prevents deletions.

It is unclear why the (CGG•CCG) and (GAA•TTC) sequences cloned in plasmids did not respond to the mutations in NER in our system. It is possible that these TRS may be too short and/or that some different structural properties of (CGG•CCG) and (GAA•TTC) (21,58,59) do not trigger the repair in E.coli. Other prior investigations have revealed the structural uniqueness of (CTG•CAG) relative to the other TRS (21,60).

We conclude that the binding of the UvrA protein to the (CTG) hairpins formed on the lagging-strand template removes its secondary structure and prevents deletions. Preferential formation of hairpins on Okazaki fragments is also believed to result in the expansion of long (CTG•CAG) repeats (5). Therefore, we speculate that these hairpins can be removed by human nucleotide excision proteins thereby stabilizing long (CTG•CAG) tracts and thus prevent expansions.

Acknowledgements

We thank A. Pluciennik and R. R. Iyer for helpful discussions. This work was supported by grants from the NIH (GM52982) and the R. A. Welch Foundation.

References

1

,

Genetic Instabilities and Hereditary Neurological Diseases

,

1998

San Diego, CA

Academic Press

2

,

Annu. Rev. Neurosci.

,

1996

, vol.

19

(pg.

79

-

107

)

3

,

J. Biol. Chem.

,

1998

, vol.

273

(pg.

19532

-

19541

)

4

,

Mol. Cell. Biol.

,

1997

, vol.

17

(pg.

2090

-

2098

)

5

,

Nature Genet.

,

1995

, vol.

10

(pg.

213

-

218

)

6

,

J. Mol. Biol.

,

1996

, vol.

258

(pg.

543

-

547

)

7

,

Nature Genet.

,

1994

, vol.

6

(pg.

114

-

116

)

8

,

J. Biol. Chem.

,

1996

, vol.

271

(pg.

2875

-

2878

)

9

,

Mol. Cell. Biol.

,

1996

, vol.

16

(pg.

6617

-

6622

)

10

,

Nature Genet.

,

1997

, vol.

17

(pg.

298

-

304

)

11

,

Science

,

1998

, vol.

279

(pg.

853

-

856

)

12

,

Am. J. Med. Genet.

,

1996

, vol.

64

(pg.

226

-

233

)

13

,

Science

,

1993

, vol.

259

(pg.

809

-

813

)

14

,

J. Med. Genet.

,

1997

, vol.

34

(pg.

924

-

926

)

15

,

Hum. Mol. Genet.

,

1994

, vol.

3

(pg.

1823

-

1927

)

16

,

Am. J. Med. Genet.

,

1996

, vol.

64

(pg.

287

-

292

)

17

,

Proc. Natl Acad. Sci. USA

,

1995

, vol.

92

(pg.

11019

-

11023

)

18

,

Nucleic Acids Res.

,

1997

, vol.

25

(pg.

2861

-

2868

)

19

,

J. Mol. Biol.

,

1996

, vol.

264

(pg.

82

-

96

)

20

,

J. Mol. Biol.

,

1996

, vol.

258

(pg.

614

-

626

)

21

,

J. Biol. Chem.

,

1996

, vol.

271

(pg.

16773

-

16783

)

22

,

J. Bacteriol.

,

1996

, vol.

178

(pg.

5042

-

5044

)

23

,

J. Biol. Chem.

,

1996

, vol.

271

(pg.

1853

-

1856

)

24

,

Hum. Mol. Genet.

,

1997

, vol.

6

(pg.

349

-

355

)

25

,

Nature Genet.

,

1997

, vol.

15

(pg.

193

-

196

)

26

,

Genomics

,

1996

, vol.

36

(pg.

47

-

53

)

27

,

Genetic Instabilities and Hereditary Neurological Diseases

,

1998

San Diego, CA

Academic Press

(pg.

509

-

528

)

28

,

Nature

,

1994

, vol.

369

(pg.

258

-

261

)

29

,

Science

,

1994

, vol.

263

(pg.

1625

-

1629

)

30

,

Science

,

1994

, vol.

266

(pg.

814

-

816

)

31

,

J. Biol. Chem.

,

1994

, vol.

269

(pg.

14367

-

14370

)

32

,

Curr. Biol.

,

1995

, vol.

5

(pg.

382

-

395

)

33

,

Science

,

1995

, vol.

266

(pg.

1959

-

1960

)

34

,

Science

,

1995

, vol.

268

(pg.

738

-

740

)

35

,

Genes Dev.

,

1996

, vol.

10

(pg.

407

-

420

)

36

,

Annu. Rev. Biochem.

,

1996

, vol.

65

(pg.

101

-

133

)

37

,

Nature

,

1997

, vol.

387

(pg.

929

-

931

)

38

,

Mol. Cell. Biol.

,

1997

, vol.

17

(pg.

2851

-

2858

)

39

,

Nucleic Acids Res.

,

1997

, vol.

25

(pg.

806

-

813

)

40

,

Hum. Genet.

,

1996

, vol.

98

(pg.

151

-

157

)

41

,

DNA Repair and Mutagenesis

,

1995

Washington, DC

ASM Press

42

,

The Metabolic Basis of Inherited Diseases

,

1989

, vol.

2

New York, NY

McGraw-Hill

(pg.

2949

-

2971

)

43

,

Proc. Natl Acad. Sci. USA

,

1994

, vol.

91

(pg.

12213

-

12217

)

44

,

Annu. Rev. Biochem.

,

1996

, vol.

65

(pg.

43

-

81

)

45

,

J. Biol. Chem.

,

1996

, vol.

271

(pg.

21462

-

21470

)

46

,

J. Bacteriol.

,

1990

, vol.

172

(pg.

6568

-

6572

)

47

,

J. Biol. Chem.

,

1998

, vol.

273

(pg.

14588

-

14595

)

48

,

Molecular Cloning: A Laboratory Manual

,

1989

2nd Ed.

Cold Spring Harbor, NY

Cold Spring Harbor Laboratory Press

49

,

Nucleic Acids Res.

,

1995

, vol.

23

(pg.

1050

-

1059

)

50

,

Nucleic Acids Res.

,

1995

, vol.

23

(pg.

4303

-

4311

)

51

,

Nucleic Acids Res.

,

1995

, vol.

23

(pg.

4055

-

4057

)

52

,

J. Mol. Biol.

,

1996

, vol.

264

(pg.

323

-

336

)

53

,

Nucleic Acids Res.

,

1996

, vol.

24

(pg.

1992

-

1998

)

54

,

Genetics

,

1966

, vol.

53

(pg.

1119

-

1136

)

55

,

Science

,

1993

, vol.

260

(pg.

53

-

58

)

56

,

Science

,

1994

, vol.

266

(pg.

1957

-

1958

)

57

,

Annu. Rev. Biochem.

,

1996

, vol.

65

(pg.

15

-

42

)

58

,

J. Biol. Chem.

,

1997

, vol.

272

(pg.

16783

-

16792

)

59

,

J. Mol. Biol.

,

1996

, vol.

263

(pg.

511

-

516

)

60

,

Science

,

1994

, vol.

265

(pg.

669

-

671

)

61

,

Science

,

1994

, vol.

266

(pg.

1954

-

1956

)

62

,

J. Biol. Chem.

,

1995

, vol.

270

(pg.

15915

-

15918

)

63

,

Genetic Instabilities and Hereditary Neurological Diseases

,

1998

San Diego, CA

Academic Press

(pg.

623

-

646

)

64

,

Annu. Rev. Biochem.

,

1996

, vol.

65

(pg.

635

-

692

)

65

,

Science

,

1989

, vol.

246

(pg.

358

-

363

)

66

,

DNA Structure and Function

,

1994

San Diego, CA

Academic Press

(pg.

95

-

133

)

67

,

J. Biol. Chem.

,

1997

, vol.

272

(pg.

16793

-

16797

)

68

,

Genetic Instabilities and Hereditary Neurological Diseases

,

1998

San Diego, CA

Academic Press

(pg.

467

-

484

)

69

,

Biochemistry

,

1991

, vol.

30

(pg.

4432

-

4443

)

70

,

Proc. Natl Acad. Sci. USA

,

1982

, vol.

79

(pg.

988

-

992

)

71

,

J. Biol. Chem.

,

1996

, vol.

271

(pg.

30672

-

30676

)

72

,

Nature

,

1985

, vol.

313

(pg.

154

-

156

)

73

,

Quart. Rev. Biophys.

,

1993

, vol.

26

(pg.

131

-

175

)

74

,

J. Mol. Biol.

,

1998

, vol.

275

(pg.

405

-

411

)

75

,

Nucleic Acids Res.

,

1998

, vol.

26

(pg.

816

-

823

)

76

,

J. Biol. Chem.

,

1995

, vol.

270

(pg.

27014

-

27021

)

77

,

J. Biol. Chem.

,

1997

, vol.

272

(pg.

16798

-

16806

)

78

,

Science

,

1996

, vol.

271

(pg.

802

-

805

)

79

,

Proc. Natl Acad. Sci. USA

,

1992

, vol.

89

(pg.

3300

-

3304

)

80

,

J. Biol. Chem.

,

1997

, vol.

272

(pg.

28906

-

28911

)

© 1999 Oxford University Press

I agree to the terms and conditions. You must accept the terms and conditions.

Submit a comment

Name

Affiliations

Comment title

Comment

You have entered an invalid code

Thank you for submitting a comment on this article. Your comment will be reviewed and published at the journal's discretion. Please check for further notifications by email.

Citations

Views

Altmetric

Metrics

Total Views 460

297 Pageviews

163 PDF Downloads

Since 2/1/2017

Citations

72 Web of Science

×

Email alerts

Citing articles via

• Latest

• Most Read

• Most Cited

More from Oxford Academic