Environmental stress induces trinucleotide repeat mutagenesis in human cells.
ABSTRACT: The dynamic mutability of microsatellite repeats is implicated in the modification of gene function and disease phenotype. Studies of the enhanced instability of long trinucleotide repeats (TNRs)-the cause of multiple human diseases-have revealed a remarkable complexity of mutagenic mechanisms. Here, we show that cold, heat, hypoxic, and oxidative stresses induce mutagenesis of a long CAG repeat tract in human cells. We show that stress-response factors mediate the stress-induced mutagenesis (SIM) of CAG repeats. We show further that SIM of CAG repeats does not involve mismatch repair, nucleotide excision repair, or transcription, processes that are known to promote TNR mutagenesis in other pathways of instability. Instead, we find that these stresses stimulate DNA rereplication, increasing the proportion of cells with >4 C-value (C) DNA content. Knockdown of the replication origin-licensing factor CDT1 eliminates both stress-induced rereplication and CAG repeat mutagenesis. In addition, direct induction of rereplication in the absence of stress also increases the proportion of cells with >4C DNA content and promotes repeat mutagenesis. Thus, environmental stress triggers a unique pathway for TNR mutagenesis that likely is mediated by DNA rereplication. This pathway may impact normal cells as they encounter stresses in their environment or during development or abnormal cells as they evolve metastatic potential.
Project description:Multiple pathways modulate the dynamic mutability of trinucleotide repeats (TNRs), which are implicated in neurodegenerative disease and evolution. Recently, we reported that environmental stresses induce TNR mutagenesis via stress responses and rereplication, with more than 50% of mutants carrying deletions or insertions-molecular signatures of DNA double-strand break repair. We now show that knockdown of alt-nonhomologous end joining (alt-NHEJ) components-XRCC1, LIG3, and PARP1-suppresses stress-induced TNR mutagenesis, in contrast to the components of homologous recombination and NHEJ, which have no effect. Thus, alt-NHEJ, which contributes to genetic mutability in cancer cells, also plays a novel role in environmental stress-induced TNR mutagenesis.
Project description:The expansion of a CAG trinucleotide repeat (TNR) sequence has been linked to several neurological disorders, for example, Huntington's disease (HD). In HD, healthy individuals have 5-35 CAG repeats. Those with 36-39 repeats have the premutation allele, which is known to be prone to expansion. In the disease state, greater than 40 repeats are present. Interestingly, the formation of non-B DNA conformations by the TNR sequence is proposed to contribute to the expansion. Here we provide the first structural and thermodynamic analysis of a premutation length TNR sequence. Using chemical probes of nucleobase accessibility, we found that similar to (CAG)(10), the premutation length sequence (CAG)(36) forms a stem-loop hairpin and contains a hot spot for DNA damage. Additionally, calorimetric analysis of a series of (CAG)(n) sequences, that includes repeat tracts in both the healthy and premutation ranges, reveal that thermodynamic stability increases linearly with the number of repeats. Based on these data, we propose that while non-B conformations can be formed by TNR tracts found in both the healthy and premutation allele, only sequences containing at least 36 repeats have sufficient thermodynamic stability to contribute to expansion.
Project description:Trinucleotide repeats (TNRs) undergo high frequency mutagenesis to cause at least 15 neurodegenerative diseases. To understand better the molecular mechanisms of TNR instability in cultured cells, a new genetic assay was created using a shuttle vector. The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is dependent on TNR length. The vector harbors the SV40 ori and large T antigen gene, allowing portability between primate cell lines. The shuttle vector is propagated in cultured cells, then recovered and analyzed in yeast using selection for reporter gene expression. We show that (CAG*CTG)25-33 contracts at frequencies as high as 1% in 293T and 293 human cells and in COS-1 monkey cells, provided that the plasmid undergoes replication. Hairpin-forming capacity of the repeat sequence stimulated contractions. Evidence for a threshold was observed between 25 and 33 repeats in COS-1 cells, where contraction frequencies increased sharply (up 720%) over a narrow range of repeat lengths. Expression of the mismatch repair protein Mlh1 does not correlate with repeat instability, suggesting contractions are independent of mismatch repair in our system. Together, these findings recapitulate certain features of human genetics and therefore establish a novel cell culture system to help provide new mechanistic insights into CAG*CTG repeat instability.
Project description:Expansion of trinucleotide repeats (TNRs) is responsible for a number of human neurodegenerative disorders. The molecular mechanisms that underlie TNR instability in humans are not clear. Based on results from model systems, several mechanisms for instability have been proposed, all of which focus on the ability of TNRs to form alternative structures during normal DNA transactions, including replication, DNA repair and transcription. These abnormal structures are thought to trigger changes in TNR length. We have previously shown that transcription-induced TNR instability in cultured human cells depends on several genes known to be involved in transcription-coupled nucleotide excision repair (NER). We hypothesized that NER normally functions to destabilize expanded TNRs. To test this hypothesis, we bred an Xpa null allele, which eliminates NER, into the TNR mouse model for spinocerebellar ataxia type 1 (SCA1), which carries an expanded CAG repeat tract at the endogenous mouse Sca1 locus. We find that Xpa deficiency does not substantially affect TNR instability in either the male or female germline; however, it dramatically reduces CAG repeat instability in neuronal tissues-striatum, hippocampus and cerebral cortex-but does not alter CAG instability in kidney or liver. The tissue-specific effect of Xpa deficiency represents a novel finding; it suggests that tissue-to-tissue variation in CAG repeat instability arises, in part, by different underlying mechanisms. These results validate our original findings in cultured human cells and suggest that transcription may induce NER-dependent TNR instability in neuronal tissues in humans.
Project description:Instability of (CTG) x (CAG) microsatellite trinucleotide repeat (TNR) sequences is responsible for more than a dozen neurological or neuromuscular diseases. TNR instability during DNA synthesis is thought to involve slipped-strand or hairpin structures in template or nascent DNA strands, although direct evidence for hairpin formation in human cells is lacking. We have used targeted recombination to create a series of isogenic HeLa cell lines in which (CTG) x (CAG) repeats are replicated from an ectopic copy of the Myc (also known as c-myc) replication origin. In this system, the tendency of chromosomal (CTG) x (CAG) tracts to expand or contract was affected by origin location and the leading or lagging strand replication orientation of the repeats, and instability was enhanced by prolonged cell culture, increased TNR length and replication inhibition. Hairpin cleavage by synthetic zinc finger nucleases in these cells has provided the first direct evidence for the formation of hairpin structures during replication in vivo.
Project description:(CTG)(n) · (CAG)(n) trinucleotide repeat (TNR) expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene causes myotonic dystrophy type 1. However, a direct link between TNR instability, the formation of noncanonical (CTG)(n) · (CAG)(n) structures, and replication stress has not been demonstrated. In a human cell model, we found that (CTG)(45) · (CAG)(45) causes local replication fork stalling, DNA hairpin formation, and TNR instability. Oligodeoxynucleotides (ODNs) complementary to the (CTG)(45) · (CAG)(45) lagging-strand template eliminated DNA hairpin formation on leading- and lagging-strand templates and relieved fork stalling. Prolonged cell culture, emetine inhibition of lagging-strand synthesis, or slowing of DNA synthesis by low-dose aphidicolin induced (CTG)(45) · (CAG)(45) expansions and contractions. ODNs targeting the lagging-strand template blocked the time-dependent or emetine-induced instability but did not eliminate aphidicolin-induced instability. These results show directly that TNR replication stalling, replication stress, hairpin formation, and instability are mechanistically linked in vivo.
Project description:Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegeneration and cancer. However, their underlying mechanisms remain to be elucidated. Recent studies have demonstrated that CAG repeat expansions can be initiated by oxidative DNA base damage and fulfilled by base excision repair (BER), suggesting active roles for oxidative DNA damage and BER in TNR instability. Here, we provide the first evidence that oxidative DNA damage can induce CTG repeat deletions along with limited expansions in human cells. Biochemical characterization of BER in the context of (CTG)20 repeats further revealed that repeat instability correlated with the position of a base lesion in the repeat tract. A lesion located at the 5'-end of CTG repeats resulted in expansion, whereas a lesion located either in the middle or the 3'-end of the repeats led to deletions only. The positioning effects appeared to be determined by the formation of hairpins at various locations on the template and the damaged strands that were bypassed by DNA polymerase ? and processed by flap endonuclease 1 with different efficiency. Our study indicates that the position of a DNA base lesion governs whether TNR is expanded or deleted through BER.
Project description:Trinucleotide repeat (TNR) expansions cause at least 17 heritable neurological diseases, including Huntington's disease. Expansions are thought to arise from abnormal processing of TNR DNA by specific trans-acting proteins. For example, the DNA repair complex MutS? (MSH2-MSH3 heterodimer) is required in mice for on-going expansions of long, disease-causing alleles. A distinctive feature of TNR expansions is a threshold effect, a narrow range of repeat units (?30-40 in humans) at which mutation frequency rises dramatically and disease can initiate. The goal of this study was to identify factors that promote expansion of threshold-length CTG•CAG repeats in a human astrocytic cell line. siRNA knockdown of the MutS? subunits MSH2 or MSH3 impeded expansions of threshold-length repeats, while knockdown of the MutS? subunit MSH6 had no effect. Chromatin immunoprecipitation experiments indicated that MutS?, but not MutS?, was enriched at the TNR. These findings imply a direct role for MutS? in promoting expansion of threshold-length CTG•CAG tracts. We identified the class II deacetylase HDAC5 as a novel promoting factor for expansions, joining the class I deacetylase HDAC3 that was previously identified. Double knockdowns were consistent with the possibility that MutS?, HDAC3 and HDAC5 act through a common pathway to promote expansions of threshold-length TNRs.
Project description:Trinucleotide repeat (TNR) expansions and deletions are associated with human neurodegenerative diseases and prostate cancer. Recent studies have pointed to a linkage between oxidative DNA damage, base excision repair (BER) and TNR expansion, which is demonstrated by the observation that DNA polymerase β (pol β) gap-filling synthesis acts in concert with alternate flap cleavage by flap endonuclease 1 (FEN1) to mediate CAG repeat expansions. In this study, we provide the first evidence that the repair of a DNA base lesion can also contribute to CAG repeat deletions that were initiated by the formation of hairpins on both the template and the damaged strand of a continuous run of (CAG)(20) or (CAG)(25) repeats. Most important, we found that pol β not only bypassed one part of the large template hairpin but also managed to pass through almost the entire length of small hairpin. The unique hairpin bypass of pol β resulted in large and small deletions in coordination with FEN1 alternate flap cleavage. Our results provide new insight into the role of BER in modulating genome stability that is associated with human diseases.
Project description:5',8-cyclo-2'-deoxypurines (cdPus) are common forms of oxidized DNA lesions resulting from endogenous and environmental oxidative stress such as ionizing radiation. The lesions can only be repaired by nucleotide excision repair with a low efficiency. This results in their accumulation in the genome that leads to stalling of the replication DNA polymerases and poor lesion bypass by translesion DNA polymerases. Trinucleotide repeats (TNRs) consist of tandem repeats of Gs and As and therefore are hotspots of cdPus. In this study, we provided the first evidence that both (5'R)- and (5'S)-5',8-cyclo-2'-deoxyadenosine (cdA) in a CAG repeat tract caused CTG repeat deletion exclusively during DNA lagging strand maturation and base excision repair. We found that a cdA induced the formation of a CAG loop in the template strand, which was skipped over by DNA polymerase ? (pol ?) lesion bypass synthesis. This subsequently resulted in the formation of a long flap that was efficiently cleaved by flap endonuclease 1, thereby leading to repeat deletion. Our study indicates that accumulation of cdPus in the human genome can lead to TNR instability via a unique lesion bypass by pol ?.