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Molecular and Cellular Biology, September 2007, p. 6209-6217, Vol. 27, No. 17
0270-7306/07/$08.00+0     doi:10.1128/MCB.00739-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Transcription-Induced CAG Repeat Contraction in Human Cells Is Mediated in Part by Transcription-Coupled Nucleotide Excision Repair{triangledown}

Yunfu Lin and John H. Wilson*

Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, Texas 77030

Received 27 April 2007/ Returned for modification 16 May 2007/ Accepted 13 June 2007


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ABSTRACT
 
Expansions of CAG repeat tracts in the germ line underlie several neurological diseases. In human patients and mouse models, CAG repeat tracts display an ongoing instability in neurons, which may exacerbate disease symptoms. It is unclear how repeats are destabilized in nondividing cells, but it cannot involve DNA replication. We showed previously that transcription through CAG repeats induces their instability (Y. Lin, V. Dion, and J. H. Wilson, Nat. Struct. Mol. Biol. 13:179-180). Here, we present a genetic analysis of the link between transcription-induced repeat instability and nucleotide excision repair (NER) in human cells. We show that short interfering RNA-mediated knockdown of CSB, a component specifically required for transcription-coupled NER (TC-NER), and knockdowns of ERCC1 and XPG, which incise DNA adjacent to damage, stabilize CAG repeat tracts. These results suggest that TC-NER is involved in the pathway for transcription-induced CAG repeat instability. In contrast, knockdowns of OGG1 and APEX1, key components involved in base excision repair, did not affect repeat instability. In addition, repeats are stabilized by knockdown of transcription factor IIS, consistent with a requirement for RNA polymerase II (RNAPII) to backtrack from a transcription block. Repeats also are stabilized by knockdown of either BRCA1 or BARD1, which together function as an E3 ligase that can ubiquitinate arrested RNAPII. Treatment with the proteasome inhibitor MG132, which stabilizes repeats, confirms proteasome involvement. We integrate these observations into a tentative pathway for transcription-induced CAG repeat instability that can account for the contractions observed here and potentially for the contractions and expansions seen with human diseases.


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INTRODUCTION
 
Several neurodegenerative diseases in humans, including Huntington disease and a number of spinocerebellar ataxias, are characterized by the germ line expansion of CAG repeat tracts to lengths that have pathological consequences in the next generation (17, 53). For these diseases, inheritance typically displays the phenomenon of anticipation, which is the progressive worsening of the disease phenotype in subsequent generations as repeats continue to expand in each passage through the germ line. Repeat instability is not confined to the germ line, however. Commonly, the neurons affected in a repeat-associated disease (along with various other somatic cells) display an expansion-biased instability that increases with age and may exacerbate the disease symptoms (20, 53, 64). Here, we focus on the somatic instability of CAG repeats.

The mechanisms of repeat instability in somatic cells are not well defined and could vary from tissue to tissue. For neurons, which no longer divide, the ongoing instability of their repeats cannot depend on DNA replication, a process often suggested as the primary mechanism for germ line instability (20, 53). Using a selective system for repeat contractions, we recently showed that transcription destabilizes CAG repeats in human cells in a way that does not require DNA replication (39). Because the genes that contain CAG repeats are widely transcribed (40), transcription-induced instability is a promising potential mechanism for somatic repeat instability.

Transcription by itself cannot change the length of a repeat tract. As proposed for other destabilizing processes, such as replication, recombination, and repair, it is likely that transcription triggers repeat instability by exposing single strands of DNA, which allows CAG repeats to form slipped-strand duplexes with CAG hairpins on one strand and CTG hairpins on the other (55, 56). These secondary structures are thought to interfere with normal DNA repair processes or stimulate aberrant ones, ultimately leading to changes in repeat tract length. The challenge is to define the pathway—the molecular participants and the sequence of events—that leads from transcription-induced secondary structures to a tract length change.

Using short interfering RNA (siRNA) knockdowns of individual DNA repair proteins, we showed previously that transcription-induced repeat contraction in human cells involves the mismatch repair (MMR) complex MutSß (MSH2/MSH3) (39), which binds larger insertion/deletion loops in mismatched duplexes (22). Because knockdown of MutSß reduces the frequency of repeat contraction, it is the normal action of this complex that promotes repeat instability. Mice deficient for MSH2 or MSH3 also show significantly altered repeat stability, namely, a lower frequency of CAG repeat expansions in the germ line and in somatic cells (31, 43, 60, 61, 72).

We also showed that XPA, a central component in nucleotide excision repair (NER), is required for transcription-induced CAG repeat contraction in human cells (39). To determine whether the transcription-coupled subpathway of NER (TC-NER) was involved in the pathway for transcription-induced contraction of CAG repeats, we knocked down individual components using siRNAs. Our results suggest that the complete TC-NER pathway is utilized during transcription-induced CAG repeat instability. We also used siRNA knockdowns and drug treatments to probe the fate of RNA polymerase II (RNAPII). These results suggest that RNAPII arrest and degradation may be essential steps in the overall pathway that leads to tract length changes. Finally, we present a pathway for transcription-induced CAG repeat instability that can account for the repeat contractions observed here and potentially for the contractions and expansions seen with human diseases.


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MATERIALS AND METHODS
 
Cell culture and HPRT+ selection. FLAH25 cells, whose construction was described previously (39), were derived from HT1080 cells (a human fibrosarcoma cell line) that carried a nonreverting mutation in the endogenous HPRT gene. FLAH25 cells, which are hypoxanthine phosphoribosyltransferase negative (HPRT–), contain an HPRT minigene that is inactivated by insertion of a (CAG)95 repeat in the intron between exons 1 and 2 and exons 3 to 9. Transcription of the HPRT minigene, which is controlled by the pTRE-CMVmini promoter, was induced by addition of doxycycline (2 µg/ml) to the medium. Cells were incubated at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium-F-12 medium supplemented with 10% dialyzed serum and 1% nonessential amino acids (Invitrogen). HPRT+ cells were selected by plating 500,000 FLAH25 cells on 100-mm-diameter dishes in HAT medium (0.1 mM hypoxanthine, 0.4 µM aminopterin, and 16 µM thymine [Sigma]) plus doxycycline for 2 weeks, with the addition of fresh doxycycline after the first week of selection. Contraction frequencies, which were calculated as the number of HPRT+ colonies divided by the number of viable cells, are the averages of results from at least five experiments.

siRNA treatment. The siRNAs used in this study are listed in Table 1. For siRNA treatments, 100,000 FLAH25 cells were plated on a 100-mm-diameter plate on day –4. On day –3, siRNAs (Dharmacon and Invitrogen) at a total concentration of 200 nM were transfected into cells by using Oligofectamine (Invitrogen). Each specific siRNA was tested at 100 nM in the presence of 100 nM vimentin siRNA or 100 nM of a second specific siRNA. Treatments with 200 nM vimentin siRNA, which does not affect repeat contraction, served as controls. On day 0, the cells again were transfected with siRNA, and cultures then were grown in the presence or absence of doxycycline. Knockdown of target gene expression was evaluated by real-time reverse transcription-PCR (RT-PCR) on day 1 or by averaging measurements made by Western blotting on days 1, 2, and 3 (Table 2). Doxycycline induction of HPRT transcription in the presence of siRNAs was assayed by real-time RT-PCR on day 1. None of the siRNAs used here substantially altered the doxycycline-induced level of HPRT transcription or dramatically changed the rate of cell proliferation (Table 2). HPRT+ colonies were selected on day 3 by growth in HAT medium supplemented with doxycycline.


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  		TABLE 1. RT-PCR primers and siRNAs used in this study


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  		TABLE 2. siRNA knockdowns and their effects on cells

Real-time RT-PCR. About two million cells were harvested for preparation of total RNA, which was obtained using RNeasy minikits (QIAGEN). For measurements of HPRT gene expression and of target gene expression, RNA was extracted from siRNA-treated cells 1 day after doxycycline had been added. For real-time RT-PCR, 50 ng of total RNA per reaction was assayed using the SYBR green RT-PCR kit (QIAGEN). Real-time RT-PCR primers are listed in Table 1. Results were normalized to the concentration of ß-actin RNA, which also was determined by real-time RT-PCR (58). HPRT gene expression was measured relative to the RNA level in untreated cells, which was arbitrarily defined as 1. For expression of target genes, we used the RNA level in vimentin siRNA-treated cells, which was defined as 100%, as the reference (Table 2). Conditions for real-time RT-PCR were 50°C for 30 min and then 95°C for 15 min, followed by 45 cycles of 94°C for 15 s, 52°C or 55°C (depending on the primer) for 30 s, and 72°C for 30 s. The relative levels of mRNA were calculated by comparing the number of cycles (generally between 15 and 25 cycles) at which the PCR products became detectable above the basal threshold.

Western blot analysis. Cells were lysed in cell lysis buffer (2% sodium dodecyl sulfate, 300 mM Tris-Cl, pH 6.8, 10% glycerol) on ice for 20 min in the presence of proteinase inhibitors and phenylmethylsulfonyl fluoride. Samples then were cleared by centrifugation (13,000 x g for 5 min) and diluted in loading buffer for analysis. About 20 µg of protein was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis on 7.5% gels, transferred to a polyvinylidene difluoride membrane (Amersham Pharmacia), and incubated with antibodies (Santa Cruz Biotech). Immunoblots then were visualized by using the enhanced chemiluminescent system (Amersham Pharmacia).

MG132 treatment. One million cells were plated on 100-mm-diameter dishes on day –1. On days 0, 1, and 2, cells were refed with medium containing MG132 and doxycycline at the concentrations indicated above. On day 3, cells were refed with medium lacking MG132 and were grown for 1 day before being plated for HPRT+ selection.


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RESULTS
 
Involvement of the TC-NER pathway. To investigate the role of NER in transcription-induced CAG repeat contraction, we used FLAH25 cells, which allowed us to assay selectively for CAG repeat contractions, as previously described (39). These cells carry an integrated copy of the HPRT minigene, the expression of which is regulated by the Tet-ON system (Fig. 1). Upon addition of doxycycline, expression of the minigene is elevated 23-fold above the baseline (39). A (CAG)95 repeat tract located in the intron inactivates the HPRT minigene in these cells. Contraction of the CAG tract to less than 39 repeats renders the cells phenotypically HPRT+, permitting them to survive HAT selection. Induction of transcription in FLAH25 cells increases the rate of CAG contraction by 15-fold above the background level (39).


Figure 1
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  		FIG. 1. Selection assay for transcription-induced contraction of CAG repeats. Long CAG tracts in the HPRT minigene are incorporated into the mRNA, rendering the protein nonfunctional and giving the cells an HPRT– phenotype; however, short CAG tracts are not efficiently incorporated, allowing sufficient normal protein to be made to give an HPRT+ phenotype (73). The HPRT minigene, under control of the TRE-pCMVmini promoter, is turned on by addition of doxycycline, which binds to the reverse tetracycline transcription activator (rtTA) and stimulates expression.

Previously, we showed that siRNA knockdown of XPA, which is essential for NER, significantly reduced the frequency of transcription-induced CAG contractions (39). We also showed that siRNA knockdown of XPC, which is required specifically for global genome NER (GG-NER), did not affect the frequency. These results suggested that transcription-induced CAG contraction might be linked to TC-NER. We sought to test directly the involvement of TC-NER by siRNA knockdown of CSB, which is specifically required for this subpathway. Two CSB siRNAs, each of which reduced CSB expression by 61 and 65% (Table 2), significantly reduced the frequency of transcription-induced CAG repeat contraction (P < 0.001 and P < 0.0001, respectively) (Fig. 2). These results confirm that elements of TC-NER are involved in the transcription-induced pathway for CAG repeat contraction.


Figure 2
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  		FIG. 2. Effects of siRNA-mediated knockdowns on transcription-induced CAG repeat contraction. The specific siRNAs used in the experiments are designated 1 or 2. Contraction frequencies were calculated as the number of HPRT+ colonies divided by the number of viable cells, averaged over at least five independent experiments. Contraction frequencies in the presence of doxycycline are shown as black bars. Contraction frequencies in the absence of doxycycline, which are shown only for the vimentin control (white bar), were similar for all experiments (data not shown). Frequencies that are significantly different from that of the vimentin siRNA are indicated with an asterisk; specific P values are given in the text.

To determine whether the complete TC-NER pathway was utilized, we used siRNAs to knock down expression of ERCC1 and XPG, which function more distally in the pathway to introduce incisions on either side of a DNA lesion. We tested two ERCC1 siRNAs, which reduced ERCC1 expression by 63 and 75%, and two XPG siRNAs, which lowered XPG expression by 63 and 72% (Table 2). As shown in Fig. 2, siRNA knockdowns of ERCC1 and XPG significantly decreased the frequency of CAG repeat contractions (P < 0.005 and P < 0.0001, respectively). Collectively, these experiments identify CSB, XPA, ERCC1, and XPG as NER components that are involved in the pathway for transcription-induced CAG repeat contraction. The participation of CSB, but not XPC (39), indicates that it is the TC-NER pathway that is used. The further involvement of XPA, ERCC1, and XPG suggests that the entire TC-NER pathway is required for transcription-induced CAG repeat instability.

A recent study using mice suggested that 8-oxoguanine DNA glycosylase (OGG1), a damage recognition component of base excision repair (BER), initiates age-dependent CAG repeat expansions in somatic cells (30). To test for the involvement of components of BER in transcription-induced repeat instability in human cells, we used siRNAs against OGG1 and apuriniic/apyrimidinic endonuclease 1 (APEX1), two key proteins involved in repair of oxidative DNA damage. These siRNA treatments, which decreased OGG1 mRNA levels by 69 and 72% and APEX1 mRNA levels by 70 and 85% (Table 2), did not affect repeat instability (Fig. 2), suggesting that transcription-induced repeat instability in cultured human cells does not involve these BER components.

Fate of RNAPII. The involvement of TC-NER in transcription-induced CAG repeat contraction suggests that noncanonical DNA structures, such as the CTG hairpins RNAPII might encounter on the template strand in the HPRT reporter gene, have the potential to arrest RNAPII, which is thought to trigger TC-NER (21, 63). Displacement of RNAPII to permit repair proteins access to the blocking lesion may involve backtracking of RNAPII or its degradation, among other possibilities (33). Transcription factor IIS (TFIIS) promotes backtracking of stalled RNAPII and cleaves the nascent transcript to create a new 3' end that is properly positioned in the active site of the enzyme (11, 23, 69). To test the possible involvement of TFIIS in transcription-induced CAG contraction, we tested two TFIIS siRNAs, which reduced TFIIS expression by 59 and 75% (Table 2). As shown in Fig. 2, each siRNA significantly reduced the frequency of transcription-induced CAG repeat contraction (P < 0.005 and P < 0.001, respectively). These results suggest that TFIIS is an element in the pathway for transcription-induced contraction of CAG repeats.

To determine whether RNAPII degradation might be relevant to transcription-induced CAG contraction, we treated cells with 0.5 µM MG132, an inhibitor of the 20S subunit of the 26S proteasome (34). As expected, MG132 causes the accumulation of ubiquitinated proteins in FLAH25 cells (Fig. 3A). The frequency of CAG contractions decreased significantly in treated cells (Fig. 3B); however, HPRT transcription was stimulated more than 10-fold over that with doxycycline alone, a response associated with the cytomegalovirus promoter used in the Tet-ON system (4). To confirm the effects of MG132 under conditions in which HPRT transcription is similar to that with doxycycline alone, we lowered the concentration of doxycycline. As shown in Fig. 3B, under these conditions (0.5 µM MG132, 0.06 µg/ml doxycycline) the frequency of CAG contractions (2.0 x 10–6 ± 1.3 x 10–6) is significantly reduced (P < 0.001) compared to the frequency (7.6 x 10–6 ± 1.3 x 10–6) in control cells (0 µM MG132, 2.0 µg/ml doxycycline). We conclude that one or more steps in the pathway for transcription-induced CAG repeat contraction depend, directly or indirectly, on proteasomal degradation.


Figure 3
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  		FIG. 3. Effects of MG132 on transcription-induced repeat contraction. (A) Treatment with MG132 leads to accumulation of high-molecular-size, ubiquitinated proteins in FLAH25 cells, as revealed by reaction with antibody against ubiquitin. From left to right, the treatments with MG132 were 0, 0.25, 0.5, and 1.0 µM. (B) Effects of MG132 treatment on HPRT mRNA levels and on transcription-induced CAG repeat contraction. HPRT mRNA levels and repeat contraction frequencies are normalized to those values in the control (doxycycline, 2.0 µg/ml; MG132, 0 µM). From left to right, the frequencies of contraction are 7.6 x 10–6 ± 1.3 x 10–6, 4.0 x 10–6 ± 1.6 x 10–6, 1.9 x 10–6 ± 1.2 x 10–6, 2.0 x 10–6 ± 1.3 x 10–6, and 0.8 x 10–6 ± 1.0 x 10–6. Contraction frequencies that are significantly different from that of the vimentin siRNA are indicated with an asterisk; specific P values are given in the text. DOX, doxycycline.

One candidate E3 ubiquitin ligase for which RNAPII has been identified as a target is BRCA1/BARD1 (28, 29, 65, 66). We tested the potential involvement of these proteins in transcription-induced repeat contraction by using siRNAs to knock down either BRCA1 or BARD1. We tested two BRCA1 siRNAs, which reduced BRCA1 expression by 64 to 78% (Table 2), and two BARD1 siRNAS, which lowered BARD1 expression by 64 and 57% (Table 2). As shown in Fig. 2, siRNA knockdowns of BRCA1 and BARD1 significantly decreased the frequency of CAG repeat contractions (P < 0.001 and P < 0.005, respectively). These results are consistent with a role for BRCA1 and BARD1 in transcription-induced CAG repeat contractions but do not define how they are involved.

How many pathways? The results shown above and in our previous study (39) demonstrate that siRNA-mediated depletion of CSB, XPA, ERCC1, XPG, MSH2, MSH3, BRCA1, BARD1, and TFIIS each reduces CAG contraction, indicating that these nine proteins have critical roles in the pathway for transcription-induced CAG repeat contraction. CSB, XPA, ERCC1, and XPG are required for TC-NER, and MSH2 and MSH3 are key components of MMR. BRCA1/BARD1, an E3 ubiquitin ligase, and TFIIS may be involved in different ways of dealing with a stalled RNAPII, but those roles are less firmly established. A critical question is whether these proteins are involved in the same pathway for repeat contraction or in different ones. To address this question, we tested the effects of simultaneous knockdown of pairs of the four proteins XPA, MSH2, BRCA1, and TFIIS, one member of each functional group. As shown in Table 3, simultaneous knockdowns gave no greater reduction in contraction frequency than individual knockdowns. These results suggest that these four proteins and, by extension, all nine proteins function in a common pathway for transcription-induced contraction of CAG repeat tracts. Because no combination of siRNAs reduced the level of repeat contractions below 30 to 40% of the untreated levels, an additional pathway (or pathways), not defined by our experiments, also may contribute to transcription-induced repeat instability.


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  		TABLE 3. CAG contraction frequencies after siRNA-mediated double knockdowns


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DISCUSSION
 
In this study and our previous one (39), we identified nine proteins, MSH2, MSH3, CSB, XPA, ERCC1, XPG, BRCA1, BARD1, and TFIIS, the activities of which are involved in generating the CAG contractions that arise during transcription of repeat tracts in human cells. Their normal activities are implicated as causative agents of instability, because siRNA-mediated knockdowns of individual proteins decrease the frequency of repeat contraction. These proteins include elements of mismatch repair (MSH2 and MSH3), the TC-NER pathway (CSB, XPA, ERCC1, and XPG), and three proteins that may deal with stalled RNAPII (TFIIS, BRCA1, and BARD1). Results of pairwise knockdowns of these proteins suggest that all nine function in a common pathway, a pathway we refer to as the transcription-induced pathway for CAG repeat instability (39). Recent support for this pathway was obtained with a SCA3 Drosophila model, in which transcription-induced CAG expansions in the germ line were shown to be dependent on XPG, a component of NER (25).

This pathway provides a plausible mechanism for the ongoing instability of CAG repeat tracts observed in neurons, which must utilize a mechanism other than DNA replication (53). Although transcription has been shown to destabilize repeat tracts in bacteria, that mechanism is thought to involve an interaction between transcription and DNA replication (5, 44, 52, 62). We have shown previously that transcription-induced instability in human cells does not depend on DNA replication (39). In Fig. 4, we illustrate a hypothetical scheme for how these proteins might collaborate during transcription-induced repeat instability. This speculative pathway potentially could account for the repeat contractions observed in this study, as well as for the contractions and expansions seen with human diseases. This working model provides a framework for discussion of our genetic results and makes predictions that can be tested biochemically.


Figure 4
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  		FIG. 4. Speculative model for transcription-induced repeat instability. The orientation of the CAG repeats in this model—CAG repeats on the top strand, CTG repeat on the bottom—is the same as their orientation in the HPRT gene. In this diagram, RNAPII transcribes from left to right. The passage of RNAPII induces secondary structures to form in the repeat tract. Stabilization of a hairpin by MutSß stalls the next RNAPII, which triggers NER. Displacement of RNAPII, presumably involving TFIIS and BRCA1/BARD1, allows NER proteins to gain access to the hairpin and initiate repair, which can shorten or lengthen the repaired strand.

A prerequisite for a transcription-induced pathway of instability is that the repeat-containing genes be transcribed in tissues in which they are unstable. This is generally true, as illustrated by the gene causing Huntington disease, which is widely transcribed in humans, rats, and mice, with its highest expression levels in brain and testis and its lowest expression levels in liver (10, 35, 67). The level of Huntington mRNA in a particular tissue, however, does not correlate with the degree of instability (35, 67), as has been observed for some other repeat diseases as well (40). The lack of a direct correlation between transcript levels and repeat instability has been cited as an argument against a role for transcription in repeat stability (19, 38). Transcription rates, however, have not been measured in these studies, nor is it essential that repeat instability correlates with the rate of secondary-structure generation by transcription; the degree of instability may depend instead on the tissue-specific efficiency with which such abnormal structures are processed by DNA repair (40).

An untested assumption of the pathway depicted in Fig. 4 is that transcription can generate abnormal DNA structures, which are the presumed substrates for repair. Transcription through a repeat might generate abnormal DNA structures in two different ways. First, RNAPII separates the individual DNA strands as it passes through a repeat, potentially allowing them to explore alternative base-pairing arrangements. Formation of abnormal structures on the template strand seems unlikely, since it passes through a well-defined channel on RNAPII and mostly is involved in an RNA-DNA hybrid with the new transcript (18). The unpaired nontemplate strand, however, is not ordered in the crystal structure (18) and thus might be able to form a hairpin. Indeed, the cotranscriptional formation of G quartets (G4 DNA) has been observed on the nontemplate strand (13). The tendency to form alternative structures could be enhanced by the formation of transcriptional R-loops, which are extended stretches of RNA-DNA hybrid on the template strand (36). Due to the exceptional stability of rG:dC base pairs (68), R-loops tend to form when the template strand is C rich, and the template strand in CAG repeats is 33% C, which is above the genome average of 21% for human cells. R-loop formation has been observed in a region of the template strand in the ß-actin gene that is 34% C (37). If a hairpin were to form in the nontemplate strand, the template strand would be forced to compensate with a hairpin of its own when it cleared RNAPII, generating a slipped-strand duplex (55, 56).

Second, it is possible that abnormal structures form at repeats, not as a direct result of strand separation by RNAPII but indirectly as a consequence of the negative supercoiling pressure generated as RNAPII moves downstream of the repeat (41). Negative supercoiling pressure would promote opening of the duplex, allowing secondary structures to form on both strands. For Escherichia coli it has been shown that CAG repeats on plasmids become more unstable as the negative supercoiling of the DNA increases (42, 45). Finally, it should be noted that these mechanisms are not mutually exclusive; for example, topoisomerase I mutants in E. coli display a higher frequency of R-loops relative to the wild type (12, 36).

Slipped-strand structures like the one represented in Fig. 4 are reasonably stable (55). In accordance with the slightly lower thermal stability of CAG hairpins (57), the slipped-strand structures formed on the CAG strand show more single-strand character than those on the CTG strand (55). For that reason, we have shown CAG structures in Fig. 4 as loops and CTG structures as hairpins, but their configurations in cells are not defined. The more single-stranded the loop, the more easily it could branch migrate, perhaps allowing multiple small CAG loops to coalesce into a larger one.

Looped-out structures, by themselves, are unlikely to block progress of RNAPII (6), which is a powerful molecular motor that is elegantly designed to separate strands (18, 76). Both CTG and CAG loops, however, are bound by MMR proteins (51, 54), which might stabilize them sufficiently to block RNAPII. For CAG hairpins, which have been more thoroughly studied, MutSß (MSH2/MSH3) binds with a low-nanomolar Kd; moreover, its binding protects the hairpin itself, rather than the surrounding duplex, as occurs with small loops (51). A protein-stabilized hairpin, orthogonal to the direction of RNAPII movement, might present an insurmountable barrier. In Fig. 4, we show the MutSß-stabilized CTG hairpin on the template strand as the blocking lesion, because for all other well-studied examples of DNA damage, ranging from base modifications (63) to topoisomerase adducts (3), RNA polymerase arrests only at sites of damage on the template strand. A MutSß-stabilized CAG hairpin on the nontemplate strand, however, is a very large entity that might interfere with RNAPII progression.

An RNA polymerase complex stalled at the site of damage is thought to constitute the primary signal to engage TC-NER (71). A likely corollary is that RNAPII then must be displaced from the block to permit access by the DNA repair machinery (69). The most likely mechanisms for displacement in eucaryotic cells include RNAPII backtracking, chromatin remodeling to reorient RNAPII, and proteasomal degradation of RNAPII (16, 69). The reduction in CAG contractions found when we knock down TFIIS suggests that RNAPII backtracking is involved in transcription-induced CAG repeat contraction. The similar effects of proteasome inhibition by MG132 and of the knockdown of either component of the BRCA1/BARD1 E3 ubiquitin ligase suggest that degradation of RNAPII also might be part of the transcription-induced pathway for CAG repeat contraction.

The diverse roles of BRCA1 make it difficult to rule out other possible interpretations (66). In support of the proposed role, BRCA1-deficient cells have been shown to be defective for TC-NER (1), BRCA1/BARD1 can bind to RNAPII (8), BRCA1 interacts with the elongating (hyperphosphorylated) form of RNAPII (32), BRCA1/BARD1 can ubiquitinate hyperphosphorylated RNAPII (28, 29, 65), and alanine substitution at either of two key damage-responsive phosphorylation sites on BARD1 abrogates RNAPII ubiquitination (28).

If both RNAPII backtracking (TFIIS) and degradation (BRCA1/BARD1) are involved in transcription-induced CAG repeat contraction, how is it that these apparent alternatives can be involved in the same pathway, as suggested by the absence of an enhanced effect by the combined knockdown of TFIIS and BRCA1? Recruitment of TFIIS to the stalled polymerase by CSA (16) suggests a potential link between TFIIS-stimulated cleavage of the 3' end of the RNA and the TC- NER pathway. Why, then, might proteasomal degradation of RNAPII be involved in transcription-coupled repair of abnormal repeat structures? One possibility is that the binding of TFIIS is a prerequisite for the regulated ubiquitination of RNAPII by BRCA1/BARD1. A second possibility relates to the specific properties of the repeats. If RNAPII stalled at a CTG hairpin (Fig. 4) exposes single strands around the base of the hairpin, backtracking may allow additional bases to be incorporated into the hairpin. Thus, the hairpin might chase the retreating polymerase, preventing access by repair factors and triggering ubiquitination and degradation.

Once assembled at the site of the stalled RNAPII, could the NER components deal with a MutSß-stabilized CTG hairpin? Although NER is designed to repair bulky lesions, virtually all biochemical studies to date have examined repair of damaged or cross-linked bases (9, 63, 70). A variety of genetic studies of bacterial and mammalian cells suggest that NER is involved in the repair of non-B DNA structures, including intramolecular triplexes (2), intermolecular triplexes (15, 74), and CNG hairpins (24, 39, 50, 52, 75). In addition, intermolecular triplexes have been shown to bind XPA in vitro (73). Collectively, these studies suggest that aberrant DNA structures also may be subject to NER, but the details are not clear.

In Fig. 4, we have modeled repair of a CTG hairpin after that for a damaged base, with repair focused on the template strand. Binding of the core NER factors positions the endonucleases XPG and ERCC1/XPF so that they can cut on either side of the hairpin, allowing it to be released from the duplex. DNA polymerase then fills the gap, and ligase seals the remaining nick. If the repaired hairpin is in an otherwise duplex segment of DNA, as shown on the left in Fig. 4, repair will yield a bottom strand (template strand) that is shorter, generating a repeat contraction that might be detected by our assay system. On the other hand, if the repaired CTG hairpin is opposite or near a CAG loop, as shown on the right in Fig. 4, DNA polymerase could copy the loop during repair, potentially generating a bottom strand that is longer than the original; that is, an expansion, as commonly observed in neurons from mouse models and human patients (26, 27).

The final product in Fig. 4 is a repaired structure with different-length strands within the repeat tract. Such a heteroduplex has not been looked for specifically in vivo. Alternatively, the structure might be subjected to further rounds of repair. In neurons and other types of differentiated cells, both strands are actively repaired: the template strand by TC-NER and the nontemplate strand by differentiation-associated repair (46-49). It is unclear how TC-NER and differentiation-associated repair might deal with CTG- and CAG-induced structures. It also is not certain that such structures would be handled in the standard way. If nicks were introduced across from the loop on the nonlooped strand instead of adjacent to the loop, the loops might be copied as part of the repair process, giving rise to expansions (19, 40, 44).

In summary, we have identified TC-NER as a key part of the pathway for transcription-induced CAG contraction in human cells. This pathway requires functional coordination of at least nine proteins, including MMR components (MSH2/MSH3), NER components (CSB, XPA, ERCC1, and XPG), a transcription factor (TFIIS), and an E3 ubiquitin ligase (BRCA1/BARD1). We have organized these proteins into a working model that can account for current observations and that makes testable predictions. Because the genes that contain CAG repeats are widely transcribed, as are most DNA repair genes, transcription-induced instability provides an attractive mechanism for the ongoing variability of CAG repeat tracts observed in neurons, and it may contribute to instability in any tissue in which the repeat tract is transcribed, including the germ line.


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ACKNOWLEDGMENTS
 
We thank Jason Shohet and Zaowen Chen for help with real-time RT-PCR and Yi Wang and Mei Leng for help with Western blotting. We thank Vincent Dion and other members of the Wilson laboratory for helpful discussions.

This work was supported by a grant from the NIH (GM38219).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-5760. Fax: (713) 796-9438. E-mail: jwilson{at}bcm.tmc.edu Back

{triangledown} Published ahead of print on 25 June 2007. Back


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REFERENCES
 
    1
  1. Abbott, D. W., M. E. Thompson, C. Robinson-Benion, G. Tomlinson, R. A. Jensen, and J. T. Holt. 1999. BRCA1 expression restores radiation resistance in BRCA1-defective cancer cells through enhancement of transcription-coupled DNA repair. J. Biol. Chem. 274:18808-18812.[Abstract/Free Full Text]
    2. 2
  2. Bacolla, A., A. Jaworski, T. D. Connors, and R. D. Wells. 2001. Pkd1 unusual DNA conformations are recognized by nucleotide excision repair. J. Biol. Chem. 276:18597-18604.[Abstract/Free Full Text]
    3. 3
  3. Bendixen, C., B. Thomsen, J. Alsner, and O. Westergaard. 1990. Camptothecin-stabilized topoisomerase I-DNA adducts cause premature termination of transcription. Biochemistry 29:5613-5619.[CrossRef][Medline]
    4. 4
  4. Biasini, E., L. Fioriti, I. Ceglia, R. Invernizzi, A. Bertoli, R. Chiesa, and G. Forloni. 2004. Proteasome inhibition and aggregation in Parkinson's disease: a comparative study in untransfected and transfected cells. J. Neurochem. 88:545-553.[CrossRef][Medline]
    5. 5
  5. Bowater, R. P., A. Jaworski, J. E. Larson, P. Parniewski, and R. D. Wells. 1997. Transcription increases the deletion frequency of long CTG.CAG triplet repeats from plasmids in Escherichia coli. Nucleic Acids Res. 25:2861-2868.[Abstract/Free Full Text]
    6. 6
  6. Buterin, T., C. Meyer, B. Giese, and H. Naegeli. 2005. DNA quality control by conformational readout on the undamaged strand of the double helix. Chem. Biol. 12:913-922.[CrossRef][Medline]
    7. 7
  7. Chang, I. Y., M. H. Kim, H. B. Kim, D. Y. Lee, S. H. Kim, H. Y. Kim, and H. J. You. 2005. Small interfering RNA-induced suppression of ERCC1 enhances sensitivity of human cancer cells to cisplatin. Biochem. Biophys. Res. Commun. 327:225-233.[CrossRef][Medline]
    8. 8
  8. Chiba, N., and J. D. Parvin. 2002. The BRCA1 and BARD1 association with the RNA polymerase II holoenzyme. Cancer Res. 62:4222-4228.[Abstract/Free Full Text]
    9. 9
  9. Cline, S. D., and P. C. Hanawalt. 2003. Who's on first in the cellular response to DNA damage? Nat. Rev. Mol. Cell Biol. 4:361-372.[CrossRef][Medline]
    10. 10
  10. Dixon, K. T., J. A. Cearley, J. M. Hunter, and P. J. Detloff. 2004. Mouse Huntington's disease homolog mRNA levels: variation and allele effects. Gene Expr. 11:221-231.[Medline]
    11. 11
  11. Donahue, B. A., S. Yin, J. S. Taylor, D. Reines, and P. C. Hanawalt. 1994. Transcript cleavage by RNA polymerase II arrested by a cyclobutane pyrimidine dimer in the DNA template. Proc. Natl. Acad. Sci. USA 91:8502-8506.[Abstract/Free Full Text]
    12. 12
  12. Drolet, M. 2006. Growth inhibition mediated by excess negative supercoiling: the interplay between transcription elongation, R-loop formation and DNA topology. Mol. Microbiol. 59:723-730.[CrossRef][Medline]
    13. 13
  13. Duquette, M. L., P. Handa, J. A. Vincent, A. F. Taylor, and N. Maizels. 2004. Intracellular transcription of G-rich DNAs induces formation of G-loops, novel structures containing G4 DNA. Genes Dev. 18:1618-1629.[Abstract/Free Full Text]
    14. 14
  14. Fabbro, M., K. Savage, K. Hobson, A. J. Deans, S. N. Powell, G. A. McArthur, and K. K. Khanna. 2004. BRCA1-BARD1 complexes are required for p53Ser-15 phosphorylation and a G1/S arrest following ionizing radiation-induced DNA damage. J. Biol. Chem. 279:31251-31258.[Abstract/Free Full Text]
    15. 15
  15. Faruqi, A. F., H. J. Datta, D. Carroll, M. M. Seidman, and P. M. Glazer. 2000. Triple-helix formation induces recombination in mammalian cells via a nucleotide excision repair-dependent pathway. Mol. Cell. Biol. 20:990-1000.[Abstract/Free Full Text]
    16. 16
  16. Fousteri, M., W. Vermeulen, A. A. van Zeeland, and L. H. Mullenders. 2006. Cockayne syndrome A and B proteins differentially regulate recruitment of chromatin remodeling and repair factors to stalled RNA polymerase II in vivo. Mol. Cell 23:471-482.[CrossRef][Medline]
    17. 17
  17. Gatchel, J. R., and H. Y. Zoghbi. 2005. Diseases of unstable repeat expansion: mechanisms and common principles. Nat. Rev. Genet. 6:743-755.[Medline]
    18. 18
  18. Gnatt, A. L., P. Cramer, J. Fu, D. A. Bushnell, and R. D. Kornberg. 2001. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 Å resolution. Science 292:1876-1882.[Abstract/Free Full Text]
    19. 19
  19. Gomes-Pereira, M., M. T. Fortune, L. Ingram, J. P. McAbney, and D. G. Monckton. 2004. Pms2 is a genetic enhancer of trinucleotide CAG.CTG repeat somatic mosaicism: implications for the mechanism of triplet repeat expansion. Hum. Mol. Genet. 13:1815-1825.[Abstract/Free Full Text]
    20. 20
  20. Gomes-Pereira, M., and D. G. Monckton. 2006. Chemical modifiers of unstable expanded simple sequence repeats: what goes up, could come down. Mutat. Res. 598:15-34.[Medline]
    21. 21
  21. Hanawalt, P. C. 1994. Transcription-coupled repair and human disease. Science 266:1957-1958.[Free Full Text]
    22. 22
  22. Iyer, R. R., A. Pluciennik, V. Burdett, and P. L. Modrich. 2006. DNA mismatch repair: functions and mechanisms. Chem. Rev. 106:302-323.[CrossRef][Medline]
    23. 23
  23. Izban, M. G., and D. S. Luse. 1992. The RNA polymerase II ternary complex cleaves the nascent transcript in a 3'-5' direction in the presence of elongation factor SII. Genes Dev. 6:1342-1356.[Abstract/Free Full Text]
    24. 24
  24. Jaworski, A., W. A. Rosche, R. Gellibolian, S. Kang, M. Shimizu, R. P. Bowater, R. R. Sinden, and R. D. Wells. 1995. Mismatch repair in Escherichia coli enhances instability of (CTG)n triplet repeats from human hereditary diseases. Proc. Natl. Acad. Sci. USA 92:11019-11023.[Abstract/Free Full Text]
    25. 25
  25. Jung, J., and N. Bonini. 2007. CREB-binding protein modulates repeat instability in a Drosophila model for polyQ disease. Science 315:1857-1859.[Abstract/Free Full Text]
    26. 26
  26. Kennedy, L., E. Evans, C. M. Chen, L. Craven, P. J. Detloff, M. Ennis, and P. F. Shelbourne. 2003. Dramatic tissue-specific mutation length increases are an early molecular event in Huntington disease pathogenesis. Hum. Mol. Genet. 12:3359-3367.[Abstract/Free Full Text]
    27. 27
  27. Kennedy, L., and P. F. Shelbourne. 2000. Dramatic mutation instability in HD mouse striatum: does polyglutamine load contribute to cell-specific vulnerability in Huntington's disease? Hum. Mol. Genet. 9:2539-2544.[Abstract/Free Full Text]
    28. 28
  28. Kim, H. S., H. Li, M. Cevher, A. Parmelee, D. Fonseca, F. E. Kleiman, and S. B. Lee. 2006. DNA damage-induced BARD1 phosphorylation is critical for the inhibition of messenger RNA processing by BRCA1/BARD1 complex. Cancer Res. 66:4561-4565.[Abstract/Free Full Text]
    29. 29
  29. Kleiman, F. E., F. Wu-Baer, D. Fonseca, S. Kaneko, R. Baer, and J. L. Manley. 2005. BRCA1/BARD1 inhibition of mRNA 3' processing involves targeted degradation of RNA polymerase II. Genes Dev. 19:1227-1237.[Abstract/Free Full Text]
    30. 30
  30. Kovtun, I. V., Y. Liu, M. Bjoras, A. Klungland, S. H. Wilson, and C. T. McMurray. 2007. OGG1 initiates age-dependent CAG trinucleotide expansion in somatic cells. Nature 447:447-452.[CrossRef][Medline]
    31. 31
  31. Kovtun, I. V., and C. T. McMurray. 2001. Trinucleotide expansion in haploid germ cells by gap repair. Nat. Genet. 27:407-411.[CrossRef][Medline]
    32. 32
  32. Krum, S. A., G. A. Miranda, C. Lin, and T. F. Lane. 2003. BRCA1 associates with processive RNA polymerase II. J. Biol. Chem. 278:52012-52020.[Abstract/Free Full Text]
    33. 33
  33. Lainé, J. P., and J. M. Egly. 2006. When transcription and repair meet: a complex system. Trends Genet. 22:430-436.[CrossRef][Medline]
    34. 34
  34. Lee, D. H., and A. L. Goldberg. 1998. Proteasome inhibitors: valuable new tools for cell biologists. Trends Cell Biol. 8:397-403.[CrossRef][Medline]
    35. 35
  35. Li, S. H., G. Schilling, W. S. Young III, X. J. Li, R. L. Margolis, O. C. Stine, M. V. Wagster, M. H. Abbott, M. L. Franz, N. G. Ranen, et al. 1993. Huntington's disease gene (IT15) is widely expressed in human and rat tissues. Neuron 11:985-993.[CrossRef][Medline]
    36. 36
  36. Li, X., and J. L. Manley. 2006. Cotranscriptional processes and their influence on genome stability. Genes Dev. 20:1838-1847.[Abstract/Free Full Text]
    37. 37
  37. Li, X., and J. L. Manley. 2005. Inactivation of the SR protein splicing factor ASF/SF2 results in genomic instability. Cell 122:365-378.[CrossRef][Medline]
    38. 38
  38. Lia, A. S., H. Seznec, H. Hofmann-Radvanyi, F. Radvanyi, C. Duros, C. Saquet, M. Blanche, C. Junien, and G. Gourdon. 1998. Somatic instability of the CTG repeat in mice transgenic for the myotonic dystrophy region is age dependent but not correlated to the relative intertissue transcription levels and proliferative capacities. Hum. Mol. Genet. 7:1285-1291.[Abstract/Free Full Text]
    39. 39
  39. Lin, Y., V. Dion, and J. H. Wilson. 2006. Transcription promotes contraction of CAG repeat tracts in human cells. Nat. Struct. Mol. Biol. 13:179-180.[CrossRef][Medline]
    40. 40
  40. Lin, Y., V. Dion, and J. H. Wilson. 2006. Transcription and triplet repeat instability, p. 691-704. In R. Wells and T. Ashizawa (ed.), Genetic instability and neurological diseases. Elsevier, Amsterdam, The Netherlands.
    41. 41
  41. Liu, L. F., and J. C. Wang. 1987. Supercoiling of the DNA template during transcription. Proc. Natl. Acad. Sci. USA 84:7024-7027.[Abstract/Free Full Text]
    42. 42
  42. Majchrzak, M., R. P. Bowater, P. Staczek, and P. Parniewski. 2006. SOS repair and DNA supercoiling influence the genetic stability of DNA triplet repeats in Escherichia coli. J. Mol. Biol. 364:612-624.[CrossRef][Medline]
    43. 43
  43. Manley, K., T. L. Shirley, L. Flaherty, and A. Messer. 1999. Msh2 deficiency prevents in vivo somatic instability of the CAG repeat in Huntington disease transgenic mice. Nat. Genet. 23:471-473.[CrossRef][Medline]
    44. 44
  44. Mochmann, L. H., and R. D. Wells. 2004. Transcription influences the types of deletion and expansion products in an orientation-dependent manner from GAC*GTC repeats. Nucleic Acids Res. 32:4469-4479.[Abstract/Free Full Text]
    45. 45
  45. Napierala, M., A. Bacolla, and R. D. Wells. 2005. Increased negative superhelical density in vivo enhances the genetic instability of triplet repeat sequences. J. Biol. Chem. 280:37366-37376.[Abstract/Free Full Text]
    46. 46
  46. Nouspikel, T., and P. C. Hanawalt. 2002. DNA repair in terminally differentiated cells. DNA Repair (Amsterdam) 1:59-75.[CrossRef]
    47. 47
  47. Nouspikel, T., and P. C. Hanawalt. 2006. Impaired nucleotide excision repair upon macrophage differentiation is corrected by E1 ubiquitin-activating enzyme. Proc. Natl. Acad. Sci. USA 103:16188-16193.[Abstract/Free Full Text]
    48. 48
  48. Nouspikel, T., and P. C. Hanawalt. 2000. Terminally differentiated human neurons repair transcribed genes but display attenuated global DNA repair and modulation of repair gene expression. Mol. Cell. Biol. 20:1562-1570.[Abstract/Free Full Text]
    49. 49
  49. Nouspikel, T. P., N. Hyka-Nouspikel, and P. C. Hanawalt. 2006. Transcription domain-associated repair in human cells. Mol. Cell. Biol. 26:8722-8730.[Abstract/Free Full Text]
    50. 50
  50. Oussatcheva, E. A., V. I. Hashem, Y. Zou, R. R. Sinden, and V. N. Potaman. 2001. Involvement of the nucleotide excision repair protein UvrA in instability of CAG*CTG repeat sequences in Escherichia coli. J. Biol. Chem. 276:30878-30884.[Abstract/Free Full Text]
    51. 51
  51. Owen, B. A., Z. Yang, M. Lai, M. Gajek, J. D. Badger, Jr., J. J. Hayes, W. Edelmann, R. Kucherlapati, T. M. Wilson, and C. T. McMurray. 2005. (CAG)n-hairpin DNA binds to Msh2-Msh3 and changes properties of mismatch recognition. Nat. Struct. Mol. Biol. 12:663-670.[CrossRef][Medline]
    52. 52
  52. Parniewski, P., A. Bacolla, A. Jaworski, and R. D. Wells. 1999. Nucleotide excision repair affects the stability of long transcribed (CTG*CAG) tracts in an orientation-dependent manner in Escherichia coli. Nucleic Acids Res. 27:616-623.[Abstract/Free Full Text]
    53. 53
  53. Pearson, C. E., K. N. Edamura, and J. D. Cleary. 2005. Repeat instability: mechanisms of dynamic mutations. Nat. Rev. Genet. 6:729-742.[CrossRef][Medline]
    54. 54
  54. Pearson, C. E., A. Ewel, S. Acharya, R. A. Fishel, and R. R. Sinden. 1997. Human MSH2 binds to trinucleotide repeat DNA structures associated with neurodegenerative diseases. Hum. Mol. Genet. 6:1117-1123.[Abstract/Free Full Text]
    55. 55
  55. Pearson, C. E., and R. R. Sinden. 1996. Alternative structures in duplex DNA formed within the trinucleotide repeats of the myotonic dystrophy and fragile X loci. Biochemistry 35:5041-5053.[CrossRef][Medline]
    56. 56
  56. Pearson, C. E., Y. H. Wang, J. D. Griffith, and R. R. Sinden. 1998. Structural analysis of slipped-strand DNA (S-DNA) formed in (CTG)n. (CAG)n repeats from the myotonic dystrophy locus. Nucleic Acids Res. 26:816-823.[Abstract/Free Full Text]
    57. 57
  57. Petruska, J., N. Arnheim, and M. F. Goodman. 1996. Stability of intrastrand hairpin structures formed by the CAG/CTG class of DNA triplet repeats associated with neurological diseases. Nucleic Acids Res. 24:1992-1998.[Abstract/Free Full Text]
    58. 58
  58. Pfaffl, M. W. 2001. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e45.[Abstract/Free Full Text]
    59. 59
  59. Quinn, J. E., R. D. Kennedy, P. B. Mullan, P. M. Gilmore, M. Carty, P. G. Johnston, and D. P. Harkin. 2003. BRCA1 functions as a differential modulator of chemotherapy-induced apoptosis. Cancer Res. 63:6221-6228.[Abstract/Free Full Text]
    60. 60
  60. Savouret, C., E. Brisson, J. Essers, R. Kanaar, A. Pastink, H. te Riele, C. Junien, and G. Gourdon. 2003. CTG repeat instability and size variation timing in DNA repair-deficient mice. EMBO J. 22:2264-2273.[CrossRef][Medline]
    61. 61
  61. Savouret, C., C. Garcia-Cordier, J. Megret, H. te Riele, C. Junien, and G. Gourdon. 2004. MSH2-dependent germinal CTG repeat expansions are produced continuously in spermatogonia from DM1 transgenic mice. Mol. Cell. Biol. 24:629-637.[Abstract/Free Full Text]
    62. 62
  62. Schumacher, S., I. Pinet, and M. Bichara. 2001. Modulation of transcription reveals a new mechanism of triplet repeat instability in Escherichia coli. J. Mol. Biol. 307:39-49.[CrossRef][Medline]
    63. 63
  63. Scicchitano, D. A., E. C. Olesnicky, and A. Dimitri. 2004. Transcription and DNA adducts: what happens when the message gets cut off? DNA Repair (Amsterdam) 3:1537-1548.[CrossRef]
    64. 64
  64. Shelbourne, P. F., C. Keller-McGandy, W. L. Bi, S. R. Yoon, L. Dubeau, N. J. Veitch, J. P. Vonsattel, N. S. Wexler, N. Arnheim, and S. J. Augood. 2007. Triplet repeat mutation length gains correlate with cell-type specific vulnerability in Huntington disease brain. Hum. Mol. Genet. 16:1133-1142.[Abstract/Free Full Text]
    65. 65
  65. Starita, L. M., A. A. Horwitz, M. C. Keogh, C. Ishioka, J. D. Parvin, and N. Chiba. 2005. BRCA1/BARD1 ubiquitinate phosphorylated RNA polymerase II. J. Biol. Chem. 280:24498-24505.[Abstract/Free Full Text]
    66. 66
  66. Starita, L. M., and J. D. Parvin. 2006. Substrates of the BRCA1-dependent ubiquitin ligase. Cancer Biol. Ther. 5:137-141.[Medline]
    67. 67
  67. Strong, T. V., D. A. Tagle, J. M. Valdes, L. W. Elmer, K. Boehm, M. Swaroop, K. W. Kaatz, F. S. Collins, and R. L. Albin. 1993. Widespread expression of the human and rat Huntington's disease gene in brain and nonneural tissues. Nat. Genet. 5:259-265.[CrossRef][Medline]
    68. 68
  68. Sugimoto, N., S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, and M. Sasaki. 1995. Thermodynamic parameters to predict stability of RNA/DNA hybrid duplexes. Biochemistry 34:11211-11216.[CrossRef][Medline]
    69. 69
  69. Svejstrup, J. Q. 2002. Mechanisms of transcription-coupled DNA repair. Nat. Rev. Mol. Cell Biol. 3:21-29.[CrossRef][Medline]
    70. 70
  70. Tornaletti, S., and P. C. Hanawalt. 1999. Effect of DNA lesions on transcription elongation. Biochimie 81:139-146.[Medline]
    71. 71
  71. Tornaletti, S., S. M. Patrick, J. J. Turchi, and P. C. Hanawalt. 2003. Behavior of T7 RNA polymerase and mammalian RNA polymerase II at site-specific cisplatin adducts in the template DNA. J. Biol. Chem. 278:35791-35797.[Abstract/Free Full Text]
    72. 72
  72. van den Broek, W. J., M. R. Nelen, D. G. Wansink, M. M. Coerwinkel, H. te Riele, P. J. Groenen, and B. Wieringa. 2002. Somatic expansion behaviour of the (CTG)n repeat in myotonic dystrophy knock-in mice is differentially affected by Msh3 and Msh6 mismatch-repair proteins. Hum. Mol. Genet. 11:191-198.[Abstract/Free Full Text]
    73. 73
  73. Vasquez, K. M., J. Christensen, L. Li, R. A. Finch, and P. M. Glazer. 2002. Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions. Proc. Natl. Acad. Sci. USA 99:5848-5853.[Abstract/Free Full Text]
    74. 74
  74. Wang, G., M. M. Seidman, and P. M. Glazer. 1996. Mutagenesis in mammalian cells induced by triple helix formation and transcription-coupled repair. Science 271:802-805.[Abstract]
    75. 75
  75. Wang, G., and K. M. Vasquez. 2006. Non-B DNA structure-induced genetic instability. Mutat. Res. 598:103-119.[Medline]
    76. 76
  76. Zlatanova, J., W. T. McAllister, S. Borukhov, and S. H. Leuba. 2006. Single-molecule approaches reveal the idiosyncrasies of RNA polymerases. Structure 14:953-966.[Medline]

Molecular and Cellular Biology, September 2007, p. 6209-6217, Vol. 27, No. 17
0270-7306/07/$08.00+0     doi:10.1128/MCB.00739-07
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