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 originlicensing 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.stress-induced mutagenesis | trinucleotide repeats | DNA rereplication | human cells | microsatellite repeats M icrosatellite repeats-runs of one to eight nucleotides repeated in tandem-account for 3% of the DNA in the human genome (1). These repeats are remarkably dynamic, mutating at rates several orders of magnitude higher than the rates of point mutations (2, 3). Microsatellite mutations typically change the number of repeated units in the tract, adding or removing individual units, making the mutations predictable and readily reversible, in contrast to point mutations. Growing evidence implicates microsatellite repeats as modifiers of gene function, traits, and diseases.

Environmental Stress Induces Trinucleotide Repeat Mutagenesis in Human Cells by Alt-Nonhomologous End Joining Repair

“…Previously, we showed that four different environmental stresses—cold, heat, hypoxic and oxidative—induce trinucleotide repeat (TNR) mutagenesis in human cells via a pathway that involves stress response factors (SRF’s) and DNA rereplication [1]. Other pathways known to modulate TNR instability—transcription, mismatch repair, nucleotide excision repair, and base excision repair—are not involved in environmental stress-induced TNR mutagenesis, consistent with the known repression of these processes during stress [1–5].…”

“…Other pathways known to modulate TNR instability—transcription, mismatch repair, nucleotide excision repair, and base excision repair—are not involved in environmental stress-induced TNR mutagenesis, consistent with the known repression of these processes during stress [1–5]. The mechanism by which the stress response and DNA rereplication bring about TNR mutagenesis remains unclear.…”

“…The mechanism by which the stress response and DNA rereplication bring about TNR mutagenesis remains unclear. Because about 50% of the CAG tracts in these stress-induced mutants harbored indels—the molecular signature of repair of double-strand breaks (DSBs)—we asked whether a DSB repair (DSBR) pathway might process repeats during environmental stress [1]. …”

“…To knock down gene expression, we used the siRNAs listed in Table S1; each gave more than 75% mRNA knockdown. We then measured the frequency of GFP+ cells at day 3 of recovery from cold, heat, and hypoxic stress by flow cytometry [1] (Supplement). Knockdown of all the components for alt-NHEJ—PARP1, XRCC1, LIG3, and RAD50—significantly reduced the frequency of GFP+ mutants in stressed cells (Figure 1).…”

Mismatch repair enhances convergent transcription-induced cell death at trinucleotide repeats by activating ATR

“…Studies of TNR instability in model organisms—bacteria, yeast, mammals, and human cells—shed light on the spectrum of DNA metabolic processes at play in expanded repeats, including replication, transcription, DNA repair, genome wide demethylation, and rereplication [11-19]. We previously showed that transcription across long CAG repeats induces repeat instability in human cells and defined the modulating role of DNA repair factors in this process [14, 20].…”