Herpes simplex virus type 1 DNA replication is specifically required for high-frequency homologous recombination between repeated sequences.

Using an assay for recombination that measures deletion of a beta-galactosidase gene positioned between two directly repeated 350-bp sequences in plasmids transiently maintained in COS cells, we have found that replication from a simian virus 40 origin produces a high frequency of nonhomologous recombination. In contrast, plasmids replicating from a herpesvirus origin (oris) in COS cells superinfected with herpes simplex virus type 1 (HSV-1) show high levels of homologous recombination between the repeats and an enhanced recombinogenicity of the HSV-1 a sequence that is not seen during simian virus 40 replication. When the same assay was used to study recombination between 120- to 150-bp repeats in uninfected Vero cells, the level of recombination was extremely low or undetectable (< 0.03%), consistent with the fact that these repeats are smaller than the minimal efficient processing sequence for homologous recombination in mammalian cells. Recombination between these short repeats was easily measurable (0.5 to 0.8%) following HSV-1 infection, suggesting that there is an alteration of the recombination machinery. The frequency of recombination between repeats of the Uc-DR1 region, previously identified as the only segment of the HSV-1 a sequence indispensable for enhanced a-sequence recombination, was not significantly higher than that measured for other short sequences.     

Herpes simplex virus type 1 recombination: the Uc-DR1 region is required for high-level a-sequence-mediated recombination.

The a sequences of herpes simplex virus type 1 are believed to be the cis sites for inversion events that generate four isomeric forms of the viral genome. Using an assay that measures deletion of a beta-galactosidase gene positioned between two directly repeated sequences in plasmids transiently maintained in Vero cells, we had found that the a sequence is more recombinogenic than another sequence of similar size. To investigate the basis for the enhanced recombination mediated by the a sequence, we examined plasmids containing direct repeats of approximately 350 bp from a variety of sources and with a wide range of G+C content. We observed that all of these plasmids show similar recombination frequencies (3 to 4%) in herpes simplex virus type 1-infected cells. However, recombination between directly repeated a sequences occurs at twice this frequency (6 to 10%). In addition, we find that insertion of a cleavage site for an a-sequence-specific endonuclease into the repeated sequences does not appreciably increase the frequency of recombination, indicating that the presence of endonuclease cleavage sites within the a sequence does not account for its recombinogenicity. Finally, by replacing segments of the a sequence with DNA fragments of similar length, we have determined that only the 95-bp Uc-DR1 segment is indispensable for high-level a-sequence-mediated recombination.     

Recombination and deletion of sequences in shuttle vector plasmids in mammalian cells.

Shuttle vector plasmids were constructed with directly repeated sequences flanking a marker gene. African green monkey kidney (AGMK) cells were infected with the constructions, and after a period of replication, the progeny plasmids were recovered and introduced into bacteria. Those colonies with plasmids that had lost the marker gene were identified, and the individual plasmids were purified and characterized by restriction enzyme digestion. Recombination between the repeated elements generated a plasmid with a precise deletion and a characteristic restriction pattern, which distinguished the recombined molecules from those with other defects in the marker gene. Recombination among the following different sequences was measured in this assay: (i) the simian virus 40 origin and enhancer region, (ii) the AGMK Alu sequence, and (iii) a sequence from plasmid pBR322. Similar frequencies of recombination among these sequences were found. Recombination occurred more frequently in Cos1 cells than in CV1 cells. In these experiments, the plasmid population with defective marker genes consisted of the recombined molecules and of the spontaneous deletion-insertion mutants described earlier. The frequency of the latter class was unaffected by the presence of the option for recombination represented by the direct repeats. Both recombination and deletion-insertion mutagenesis were stimulated by double-strand cleavage between the repeated sequences and adjacent to the marker, and the frequency of the deletion-insertion mutants in this experiment was again independent of the presence of the direct repeats. We concluded that although recombination and deletion-insertion mutagenesis were both stimulated by double-strand cleavage, the molecules which underwent the two types of change were drawn from separate pools.     

High-frequency intermolecular homologous recombination during herpes simplex virus-mediated plasmid DNA replication

Homologous recombination is a prominent feature of herpes simplex virus (HSV) type 1 DNA replication. This has been demonstrated and traditionally studied in experimental settings where repeated sequences are present or are being introduced into a single molecule for subsequent genome isomerization. In the present study, we have designed a pair of unique HSV amplicon plasmids to examine in detail intermolecular homologous recombination (IM-HR) between these amplicon plasmids during HSV-mediated DNA replication. Our data show that IM-HR occurred at a very high frequency: up to 60% of the amplicon concatemers retrieved from virion particles underwent intermolecular homologous recombination. Such a high frequency of IM-HR required that both plasmids be replicated by HSV-mediated replication, as IM-HR events were not detected when either one or both plasmids were replicated by simian virus 40-mediated DNA replication, even with the presence of HSV infection. In addition, the majority of the homologous recombination events resulted in sequence replacement or targeted gene repair, while the minority resulted in sequence insertion. These findings imply that frequent intermolecular homologous recombination may contribute directly to HSV genome isomerization. In addition, HSV-mediated amplicon replication may be an attractive model for studying intermolecular homologous recombination mechanisms in general in a mammalian system. In this regard, the knowledge obtained from such a study may facilitate the development of better strategies for targeted gene correction for gene therapy purposes.     

Identification of herpes simplex virus type 1 genes required for origin-dependent DNA synthesis.

The herpes simplex virus (HSV) genome contains both cis- and trans-acting elements which are important in viral DNA replication. The cis-acting elements consist of three origins of replication: two copies of oriS and one copy of oriL. It has previously been shown that five cloned restriction fragments of HSV-1 DNA together can supply all of the trans-acting functions required for the replication of plasmids containing oriS or oriL when cotransfected into Vero cells (M. D. Challberg, Proc. Natl. Acad. Sci. USA, 83:9094-9098, 1986). These observations provide the basis for a complementation assay with which to locate all of the HSV sequences which encode trans-acting functions necessary for origin-dependent DNA replication. Using this assay in combination with the data from large-scale sequence analysis of the HSV-1 genome, we have now identified seven HSV genes which are necessary for transient replication of plasmids containing either oriS or oriL. As shown previously, two of these genes encode the viral DNA polymerase and single-stranded DNA-binding protein, which are known from conventional genetic analysis to be essential for viral DNA replication in infected cells. The functions of the products of the remaining five genes are unknown. We propose that the seven genes essential for plasmid replication comprise a set of genes whose products are directly involved in viral DNA synthesis.     

Herpes simplex virus: selection of origins of DNA replication.

A selection procedure was devised to study the role of cis -acting sequences at origins of DNA replication. Two regions in Herpes simplex virus oriS were examined: an AT-rich spacer sequence and a putative binding site, box III, for the origin binding protein. Plasmid libraries were generated using oligonucleotides with locally random sequences. The library, amplified in Escherichia coli , was used to transfect BHK cells followed by superinfection with HSV-1. Replicated plasmids resistant to Dpn I cleavage were amplified in E. coli. The selection scheme was repeated. Plasmids were isolated at different stages of the procedure and their replication efficiency was determined. Efficiently replicating plasmids had a high AT content in the spacer sequence as well as a low helical stability of this region. In contrast, this was not seen using the box III library. We also noted that the wild type sequence invariably dominated the library after five rounds of selection. These plasmids arose from recombination between plasmids and viral DNA. Our results imply that a large group of sequences can mechanistically serve as origins of DNA replication. In a viral system, however, where the initiation process might be rate-limiting, this potentially large group of sequences would always converge towards the most efficient replicator.     

Direct repeats of the herpes simplex virus a sequence promote nonconservative homologous recombination that is not dependent on XPF/ERCC4.

We have examined mechanisms of recombination in mammalian cells infected with herpes simplex virus type 1 (HSV-1). Amplification of plasmids containing a viral origin of replication, oriS, in cells superinfected with HSV-1 revealed that linear DNA could be efficiently converted to templates for replication. Two distinct pathways were observed: imprecise end joining and nonconservative homologous recombination. We noted that direct repeats of the viral a sequence promoted efficient nonconservative homologous recombination in BHK cells as well as human repair-proficient 1BR.3N cells and xeroderma pigmentosum group F (XP-F) cells. The reaction gave rise to functional a sequences supporting the formation of defective viruses. It did not seem to proceed by single-strand annealing since it occurred in the absence of XPF/ERCC4, the mammalian homolog of the Rad1 endonuclease from Saccharomyces cerevisiae. In contrast, direct repeats of a 161-bp nonviral sequence did not take part in nonconservative homologous recombination in XP-F cells. Our results suggest that homologous recombination may be involved in the circularization of viral genomes. Furthermore, they demonstrate that amplification of recombination products supported by HSV-1 allows a direct examination of pathways for double-strand-break repair in human cells.     

Long CTG.CAG repeat sequences markedly stimulate intramolecular recombination

Previous studies have shown that homologous recombination is a powerful mechanism for generation of massive instabilities of the myotonic dystrophy CTG.CAG sequences. However, the frequency of recombination between the CTG.CAG tracts has not been studied. Here we performed a systematic study on the frequency of recombination between these sequences using a genetic assay based on an intramolecular plasmid system in Escherichia coli. The rate of intramolecular recombination between long CTG.CAG tracts oriented as direct repeats was extraordinarily high; recombinants were found with a frequency exceeding 12%. Recombination occurred in both RecA(+) and RecA(-) cells but was approximately 2-11 times higher in the recombination proficient strain. Long CTG.CAG tracts recombined approximately 10 times more efficiently than non-repeating control sequences of similar length. The recombination frequency was 60-fold higher for a pair of (CTG.CAG)(165) tracts compared with a pair of (CTG.CAG)(17) sequences. The CTG.CAG sequences in orientation II (CTG repeats present on a lagging strand template) recombine approximately 2-4 times more efficiently than tracts of identical length in the opposite orientation relative to the origin of replication. This orientation effect implies the involvement of DNA replication in the intramolecular recombination between CTG.CAG sequences. Thus, long CTG.CAG tracts are hot spots for genetic recombination.     

Efficient Herpes Simplex Virus 1 Replication Requires Cellular ATR Pathway Proteins

Herpes simplex virus 1 (HSV-1) is a double-stranded DNA virus that replicates in the nucleus of the host cell and is known to interact with several components of the cellular DNA-damage-signaling machinery. We have previously reported that the DNA damage response kinase, ATR, is specifically inactivated in HSV-1-infected cells. On the other hand, we have also shown that ATR and its scaffolding protein, ATRIP, are recruited to viral replication compartments, where they play beneficial roles during HSV-1 replication. In order to better understand this apparent discrepancy, we tested the hypothesis that some of the components of the ATR pathway may exert an antiviral effect on infection. In fact, we learned that all 10 of the canonical ATR pathway proteins are stable in HSV-infected cells and are recruited to viral replication compartments; furthermore, short hairpin RNA (shRNA) knockdown shows that several, including ATRIP, RPA70, TopBP1, Claspin, and CINP, are required for efficient HSV-1 replication. We also determined that activation of the ATR kinase prior to infection did not affect virus yield but did result in reduced levels of recombination between coinfecting viruses. Together, these data suggest that ATR pathway proteins are not antiviral per se but that activation of ATR signaling may have negative consequences during viral replication, such as inhibiting recombination.     

Recombination during early herpes simplex virus type 1 infection is mediated by cellular proteins

Homologous recombination was examined in cells infected with herpes simplex virus type I. Circular and linear DNA with directly repeated sequences was introduced as recombination substrates into cells. Recombination was measured either by origin-dependent amplification of recombination products or by recombination-dependent expression of luciferase from a disrupted gene. Homologous recombination in baby hamster kidney cells converted linear DNA to circular templates for DNA replication and luciferase expression in the complete absence of virus. The products of homologous recombination were efficiently amplified by the viral replication apparatus. The efficiency of recombination was dependent on the structure of the substrate as well as the cell type. Linear DNA with the direct repeats at internal positions failed to recombine in Balb/c 3T3 cells and induced p53-dependent apoptosis. In contrast, linear DNA with directly repeated sequences precisely at the ends recombined and replicated in 3T3 cells. Homologous recombination in baby hamster kidney cells did not depend on the position of the repeated sequences. We conclude that homologous recombination is independent of viral gene functions and that it is likely to be carried out by cellular proteins. We suggest that homologous recombination between directly repeated sequences in the linear herpes simplex virus type 1 chromosome may help to avoid p53-dependent apoptosis and to promote viral DNA replication.     

Deletion mutagenesis independent of recombination in bacteriophage T7.

Deletion between directly repeated DNA sequences in bacteriophage T7-infected Escherichia coli was examined. The phage ligase gene was interrupted by insertion of synthetic DNA designed so that the inserts were bracketed by 10-bp direct repeats. Deletion between the direct repeats eliminated the insert and restored the ability of the phage to make its own ligase. The deletion frequency of inserts of 85 bp or less was of the order of 10(-6) deletions per replication. The deletion frequency dropped sharply in the range between 85 and 94 bp and then decreased at a much lower rate over the range from 94 to 900 bp. To see whether a deletion was predominantly caused by intermolecular recombination between the leftmost direct repeat on one chromosome and the rightmost direct repeat on a distinct chromosome, genetic markers were introduced to the left and right of the insert in the ligase gene. Short deletions of 29 bp and longer deletions of approximately 350 bp were examined in this way. Phage which underwent deletion between the direct repeats had the same frequency of recombination between the left and right flanking markers as was found in controls in which no deletion events took place. These data argue against intermolecular recombination between direct repeats as a major factor in deletion in T7-infected E. coli.     

Knockdown of DNA ligase IV/XRCC4 by RNA interference inhibits herpes simplex virus type I DNA replication

Herpes simplex virus has a linear double-stranded DNA genome with directly repeated terminal sequences needed for cleavage and packaging of replicated DNA. In infected cells, linear genomes rapidly become endless. It is currently a matter of discussion whether the endless genomes are circles supporting rolling circle replication or arise by recombination of linear genomes forming concatemers. Here, we have examined the role of mammalian DNA ligases in the herpes simplex virus, type I (HSV-1) life cycle by employing RNA interference (RNAi) in human 1BR.3.N fibroblasts. We find that RNAi-mediated knockdown of DNA ligase IV and its co-factor XRCC4 causes a hundred-fold reduction of virus yield, a small plaque phenotype, and reduced DNA synthesis. The effect is specific because RNAi against DNA ligase I or DNA ligase III fail to reduce HSV-1 replication. Furthermore, RNAi against DNA ligase IV and XRCC4 does not affect replication of adenovirus. In addition, high multiplicity infections of HSV-1 in human DNA ligase IV-deficient cells reveal a pronounced delay of production of infectious virus. Finally, we demonstrate that formation of endless genomes is inhibited by RNAi-mediated depletion of DNA ligase IV and XRCC4. Our results suggests that DNA ligase IV/XRCC4 serves an important role in the replication cycle of herpes viruses and is likely to be required for the formation of the endless genomes early during productive infection.     

Homologous and nonhomologous recombination in monkey cells.

Though recombinational events are important for the proper functioning of most cells, little is known about the frequency and mechanisms of recombination in mammalian cells. We have used simian virus 40 (SV40)-pBR322 hybrid plasmids constructed in vitro as substrates to detect and quantitate intramolecular homologous and nonhomologous recombination events in cultured monkey cells. Excision of wild-type or defective SV40 DNAs by recombination from these plasmids was scored by the viral plaque assay, in either the absence or the presence of DNA from a temperature-sensitive helper virus. Several independent products of homologous and nonhomologous recombination have been isolated and characterized at the DNA sequence level. We find that neither DNA replication of the recombination substrate nor SV40 large T antigen is essential for either homologous or nonhomologous recombination involving viral or pBR322 sequences.     

Deoxyribonucleoside triphosphate pools in synchronized human cells infected with herpes simplex virus types 1 and 2.

Deoxyribonucleoside triphosphate pools in uninfected and herpes simplex virus type 1 (HSV-1)- and HSV-2-infected KB cells were analyzed to determine whether ribonucleotide reductase functions in vivo in the presence and absence of thymidine (TdR). Previously we showed that HSV-2 replication was inhibited in KB cells blocked in their capacity to synthesize DNA by TdR. HSV-1 replication was not inhibited under these conditions. Both HSV-1 and HSV-2 induced an altered ribonucleotide reductase resistant to dTTP inhibition. Thus, the block to HSV-2 replication apparently was not at the level of reductase. However, the in vitro activity of the enzyme does not necessarily correspond to intracellular conditions. In TdR-blocked HSV-2-infected cells, we found that, while dTTP levels remained high, dCTP concentrations increased. In contrast, KB cells blocked by TdR showed increased dTTP but decreased dCTP levels. We conclude that the HSV-2 enzyme is functional in vivo and that TdR inhibits viral replication by a mechanism other than depletion of dCTP. Infection of KB cells with HSV-1 or HSV-2 altered both dATP and dGTP levels in the presence or absence of TdR. Inhibition of viral replication was not explained by changes in these pools. We suggest that, during infection, HSV-1 induces a virus function(s) not related to reductase which is resistant to TdR, whereas the corresponding HSV-2 function is sensitive. Our evidence shows that the TdR-sensitive function is not in the pathways leading to deoxyribonucleoside triphosphate and may occur at the level of DNA replication.     

Herpes simplex type 1 activates glycolysis through engagement of the enzyme 6-phosphofructo-1-kinase (PFK-1)

Viruses such as HIV, HCV, Mayaro and HCMV affect cellular metabolic pathways, including glycolysis. Although some studies have suggested that the inhibition of glycolysis affects HSV-1 replication and that HSV-1-infected eyes have increased lactate production, the mechanisms by which HSV-1 induces glycolysis have never been investigated in detail. In this study, we observed an increase in glucose uptake, lactate efflux and ATP content in HSV-1-infected cells. HSV-1 triggered a MOI-dependent increase in the activity of phosphofructokinase-1 (PFK-1), a key rate-limiting enzyme of the glycolytic pathway. After HSV-1 infection, we observed increased PFK-1 expression, which increased PFK-1 total activity, and the phosphorylation of this enzyme at serine residues. HSV-1-induced glycolysis was associated with increased ATP content, and these events were critical for viral replication. In summary, our results suggest that HSV-1 triggers glycolysis through a different mechanism than other herpesviruses, such as HCMV. Thus, this study contributes to a better understanding of HSV-1 pathogenesis and provides insights into novel targets for antiviral therapy. HIGHLIGHTS: ?HSV-1 activates glycolysis by PFK-1 activation. ?In HSV-1-infected cells PFK-1 synthesis is up-regulated and phosphorylated at serine residues. ?PFK-1 knockdown impairs HSV-1 replication. ?HSV-1-mediated glycolysis activation increases ATP content.     

Equimolar generation of the four possible arrangements of adjacent L components in herpes simplex virus type 1 replicative intermediates.

Herpes simplex virus type 1 (HSV-1) replication generates high-molecular-weight intermediates containing branched DNA and concatemers carrying adjacent genomes with inverted L components. We have studied replicative intermediates generated by (i) wild-type HSV-1; (ii) 5dl1.2, an ICP27 null mutant which fails to synthesize normal amounts of DNA and late proteins; (iii) RBMu3, a mutant containing a deletion in the inverted repeats which fails to generate genomic isomers; and (iv) amplicon plasmids and vectors which contain no inverted sequences. Replication intermediates were analyzed by pulsed-field gel electrophoresis, after restriction enzyme digestion of infected-cell DNA, followed by blot hybridization. DNA fragments were statistically quantified after phosphorimaging. We observed that (i) the four possible configurations of L components of two adjacent genomes in the concatemers are present at equimolar amounts at any time during virus replication, (ii) ICP27 is not required for inversions or for branched DNA to occur, and (iii) replication intermediates of both RBMu3 mutant and amplicon plasmids or vectors do contain branched structures, although the concatemers they generate contain no inversions. These data indicate that inversions are generated by a mechanism intrinsically linked to virus DNA replication, most likely homologous recombination between inverted repeats. Branched structures are detected in all replicating molecules, including those that do not invert, suggesting that they are constitutively linked to virus DNA synthesis. Our results are consistent with the notion that the four HSV-1 genomic isomers are generated by alternative cleavage frames of replication concatemers containing equimolar amounts of L-component inversions.     

Breakage--reunion and copy choice mechanisms of recombination between short homologous sequences.

To study recombination between short homologous sequences in Escherichia coli we constructed plasmids composed of the pBR322 replicon, M13 replication origin and a recombination unit inserted within and inactivating a gene encoding chloramphenicol resistance. The unit was composed of short direct repeats (9, 18 or 27 bp) which flanked inverted repeats (0, 8 or 308 bp) and a gene encoding kanamycin resistance. Recombination between direct repeats restored a functional chloramphenicol resistance gene, and could be detected by a simple phenotype test. The plasmids replicated in a double-stranded form, using the pBR322 replicon, and generated single-stranded DNA when the M13 replication origin was activated. The frequency of chloramphenicol-resistant cells was low (10(-8)-10(-4] when no single-stranded DNA was synthesized but increased greatly (to 100%) after induction of single-stranded DNA synthesis. Recombination between 9 bp direct repeats entailed no transfer of DNA from parental to recombinant plasmids, whereas recombination between 18 or 27 bp repeats entailed massive transfer. The presence or length of inverted repeats did not alter the pattern of DNA transfer. From these results we propose that direct repeats of 9 bp recombine by a copy choice process, while those greater than or equal to 18 bp can recombine by a breakage-reunion process. Genome rearrangements detected in many organisms often occur by recombination between sequences less than 18 bp, which suggests that they may result from copy choice recombination.     

Site-specific and illegitimate recombination in the oriV1 region of the F factor. DNA sequences involved in recombination and resolution

We have defined some of the sequences involved in high frequency recA-independent recombination at the oriV1 region of the F factor. Using a mobilization assay, we determined that plasmid pMB080, a pBR322 derivative bearing the PvuII-BamHI (F factor co-ordinates 45.43 to 46.0) fragment from the oriV1 region of F, contained all sequences necessary to undergo efficient site-specific recombination with the F derivative pOX38, which retains the oriV1 region. We constructed a series of pMB080 deletions in vitro using exonucleases S1 and Bal31. Deletions removing a ten base-pair sequence, which forms part of an inverted repeat segment located 62 base-pairs to the left of the NcoI site (45.87) within the cloned fragment, totally eliminated the recA-independent recombination reaction. Other deletions differentially affected both the frequency and stability of cointegrate molecules formed by the site-specific recombination system. The F factor oriV1 region is involved also in low-frequency recombination with several sites on pBR322 and related plasmids. We have determined the precise location of these recombination sites within oriV1 by DNA sequencing. These studies revealed that recombination always took place within an eight base-pair spacer region between the ten base-pair inverted repeats found to be important for oriV1-oriV1 interactions. We propose that the low-efficiency recombination between pBR322 and pOX38 results from the ability of the F site-specific recombination apparatus to weakly recognize and interact with sequences that bear some resemblance to the normal oriV1 recognition elements. Furthermore, we suggest, by analogy with the lambda paradigm, that the nucleotide sequences at the junctions of secondary site recombinants define at least one crossover site used during the normal site-specific recombination process.     

Endonuclease G, a candidate human enzyme for the initiation of genomic inversion in herpes simplex type 1 virus

The herpes simplex virus type 1 (HSV-1) a sequence is present as a direct repeat at the two termini of the 152-kilobase viral genome and as an inverted repeat at the junction of the two unique components L and S. During replication, the HSV-1 genome undergoes inversion of L and S, producing an equimolar mixture of the four possible isomers. Isomerization is believed to result from recombination triggered by breakage at the a sequence, a recombinational hot spot. We have identified an enzyme in HeLa cell extracts that preferentially cleaves the a sequence and have purified it to near homogeneity. Microsequencing showed it to be human endonuclease G, an enzyme with a strong preference for G+C-rich sequences. Endonuclease G appears to be the only cellular enzyme that can specifically cleave the a sequence. Endonuclease G also showed the predicted recombination properties in an in vitro recombination assay. Based on these findings, we propose that endonuclease G initiates the a sequence-mediated inversion of the L and S components during HSV-1 DNA replication.     

Long CTG.CAG repeats from myotonic dystrophy are preferred sites for intermolecular recombination

Homologous recombination was shown to enable the expansion of CTG.CAG repeat sequences. Other prior investigations revealed the involvement of replication and DNA repair in these genetic instabilities. Here we used a genetic assay to measure the frequency of homologous intermolecular recombination between two CTG.CAG tracts. When compared with non-repeating sequences of similar lengths, long (CTG.CAG)(n) repeats apparently recombine with an approximately 60-fold higher frequency. Sequence polymorphisms that interrupt the homogeneity of the CTG.CAG repeat tracts reduce the apparent recombination frequency as compared with the pure uninterrupted repeats. The orientation of the repeats relative to the origin of replication strongly influenced the apparent frequency of recombination. This suggests the involvement of DNA replication in the recombination process of triplet repeats. We propose that DNA polymerases stall within the CTG.CAG repeat tracts causing nicks or double-strand breaks that stimulate homologous recombination. The recombination process is RecA-dependent.