Mechanistic features of CAG*CTG repeat contractions in cultured cells revealed by a novel genetic assay

Abstract

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.

Coordinated Processing of 3′ Slipped (CAG)n/(CTG)n Hairpins by DNA Polymerases β and δ Preferentially Induces Repeat Expansions

“…As a major contributor to CAG/CTG repeat expansion, hairpin formation within the repeats is associated with DNA replication (10,12,13) and repair (2,14). Because DNA expansion requires DNA synthesis, DNA polymerases must play a major role in this process.…”

“…Increasing evidence suggests that hairpin formation occurs via DNA strand slippage during DNA metabolic processes that introduce DNA strand breaks within or near the repeat region (3,4,11). These processes include DNA replication (10,12,13), repair (2,14,15) and recombination (16,17). In addition to DNA strand breaks, which provide opportunities for strand slippage, these processes share a common feature in DNA synthesis, a reaction catalyzed by DNA polymerases.…”

MLH1 Founder Mutations with Moderate Penetrance in Spanish Lynch Syndrome Families

“…HCT116 [deficient for endogenous MLH1 (24)] and 293 cells were obtained from the American Type Culture Collection, resuscitated from stocks frozen at low passage within 6 months of purchase, and cultured as described (25,26). Cell lines were routinely tested by Mycoplasma presence and verified by morphology, growth curve analysis, and expression of proteins (e.g., MLH1 and PMS2).…”

Zinc-finger directed double-strand breaks within CAG repeat tracts promote repeat instability in human cells

“…Length-dependent modifiers of repeat instability are common (32)(33)(34)(35). To explore whether ZFN-induced instability depends on the length of the repeat tract, we transfected the zfGCT nuclease into APRT(CAG) 61 CHO cells and human HPRT(CAG) 68 cells.…”