The RNA export and RNA decay complexes THO and TRAMP prevent transcription-replication conflicts, DNA breaks, and CAG repeat contractions.

Expansion of structure-forming CAG/CTG repetitive sequences is the cause of several neurodegenerative disorders and deletion of repeats is a potential therapeutic strategy. Transcription-associated mechanisms are known to cause CAG repeat instability. In this study, we discovered that Thp2, an RNA export factor and member of the THO (suppressors of transcriptional defects of hpr1Δ by overexpression) complex, and Trf4, a key component of the TRAMP (Trf4/5-Air1/2-Mtr4 polyadenylation) complex involved in nuclear RNA polyadenylation and degradation, are necessary to prevent CAG fragility and repeat contractions in a Saccharomyces cerevisiae model system. Depletion of both Thp2 and Trf4 proteins causes a highly synergistic increase in CAG repeat fragility, indicating a complementary role of the THO and TRAMP complexes in preventing genome instability. Loss of either Thp2 or Trf4 causes an increase in RNA polymerase stalling at the CAG repeats and other genomic loci, as well as genome-wide transcription-replication conflicts (TRCs), implicating TRCs as a cause of CAG fragility and instability in their absence. Analysis of the effect of RNase H1 overexpression on CAG fragility, RNAPII stalling, and TRCs suggests that RNAPII stalling with associated R-loops are the main cause of CAG fragility in the thp2Δ mutants. In contrast, CAG fragility and TRCs in the trf4Δ mutant can be compensated for by RPA overexpression, suggesting that excess unprocessed RNA in TRAMP4 mutants leads to reduced RPA availability and high levels of TRCs. Our results show the importance of RNA surveillance pathways in preventing RNAPII stalling, TRCs, and DNA breaks, and show that RNA export and RNA decay factors work collaboratively to maintain genome stability.

Differential requirement of Srs2 helicase and Rad51 displacement activities in replication of hairpin-forming CAG/CTG repeats.

Trinucleotide repeats are a source of genome instability, causing replication fork stalling, chromosome fragility, and impaired repair. Specialized helicases play an important role in unwinding DNA structures to maintain genome stability. The Srs2 helicase unwinds DNA hairpins, facilitates replication, and prevents repeat instability and fragility. However, since Srs2 is a multifunctional protein with helicase activity and the ability to displace Rad51 recombinase, it was unclear which functions were required for its various protective roles. Here, using SRS2 separation-of-function alleles, we show that in the absence of Srs2 recruitment to PCNA or in helicase-deficient mutants, breakage at a CAG/CTG repeat increases. We conclude that Srs2 interaction with PCNA allows the helicase activity to unwind fork-blocking CAG/CTG hairpin structures to prevent breaks. Independently of PCNA binding, Srs2 also displaces Rad51 from nascent strands to prevent recombination-dependent repeat expansions and contractions. By 2D gel electrophoresis, we detect two different kinds of structured intermediates or joint molecules (JMs). Some JMs are Rad51-independent and exhibit properties of reversed forks, including being processed by the Exo1 nuclease. In addition, in a helicase-deficient mutant, Rad51-dependent JMs are detected, probably corresponding to recombination between sisters. These results clarify the many roles of Srs2 in facilitating replication through fork-blocking hairpin lesions.

New functions of Ctf18-RFC in preserving genome stability outside its role in sister chromatid cohesion.

Expansion of DNA trinucleotide repeats causes at least 15 hereditary neurological diseases, and these repeats also undergo contraction and fragility. Current models to explain this genetic instability invoke erroneous DNA repair or aberrant replication. Here we show that CAG/CTG tracts are stabilized in Saccharomyces cerevisiae by the alternative clamp loader/unloader Ctf18-Dcc1-Ctf8-RFC complex (Ctf18-RFC). Mutants in Ctf18-RFC increased all three forms of triplet repeat instability--expansions, contractions, and fragility--with effect over a wide range of allele lengths from 20-155 repeats. Ctf18-RFC predominated among the three alternative clamp loaders, with mutants in Elg1-RFC or Rad24-RFC having less effect on trinucleotide repeats. Surprisingly, chl1, scc1-73, or scc2-4 mutants defective in sister chromatid cohesion (SCC) did not increase instability, suggesting that Ctf18-RFC protects triplet repeats independently of SCC. Instead, three results suggest novel roles for Ctf18-RFC in facilitating genomic stability. First, genetic instability in mutants of Ctf18-RFC was exacerbated by simultaneous deletion of the fork stabilizer Mrc1, but suppressed by deletion of the repair protein Rad52. Second, single-cell analysis showed that mutants in Ctf18-RFC had a slowed S phase and a striking G2/M accumulation, often with an abnormal multi-budded morphology. Third, ctf18 cells exhibit increased Rad52 foci in S phase, often persisting into G2, indicative of high levels of DNA damage. The presence of a repeat tract greatly magnified the ctf18 phenotypes. Together these results indicate that Ctf18-RFC has additional important functions in preserving genome stability, besides its role in SCC, which we propose include lesion bypass by replication forks and post-replication repair.