Nucleotide Excision Repair/TFIIH Helicases Rad3 and Ssl2 Inhibit Short-Sequence Recombination and Ty1 Retrotransposition by Similar Mechanisms

Abstract

Eukaryotic genomes contain potentially unstable sequences whose rearrangement threatens genome structure and function. Here we show that certain mutant alleles of the nucleotide excision repair (NER)/TFIIH helicase genes RAD3 and SSL2 (RAD25) confer synthetic lethality and destabilize the Saccharomyces cerevisiae genome by increasing both short-sequence recombination and Ty1 retrotransposition. The rad3-G595R and ssl2-rtt mutations do not markedly alter Ty1 RNA or protein levels or target site specificity. However, these mutations cause an increase in the physical stability of broken DNA molecules and unincorporated Ty1 cDNA, which leads to higher levels of short-sequence recombination and Ty1 retrotransposition. Our results link components of the core NER/TFIIH complex with genome stability, homologous recombination, and host defense against Ty1 retrotransposition via a mechanism that involves DNA degradation.

Activation of a LTR-retrotransposon by telomere erosion

“…Likewise, the levels of a TyA:GFP fusion protein expressed from a chromosomal element were not significantly increased as telomeres eroded, indicating that the dramatic increase in Ty1 cDNA in est2⌬ mutants occurs by a posttranslational mechanism. Several other hypertransposition mutants exhibit a posttranslational increase in Ty1 cDNA, which has been attributed to increased cDNA stability (7,26). However, the half-life of Ty1 cDNA is at least 90 min in all wild-type strains tested to date, which is at odds with the idea that Ty1 cDNA is degraded.…”

“…To determine whether Ty1 cDNA is stabilized by the Ty1 activation pathway that is triggered by telomere erosion, we measured the half-life of Ty1 cDNA after treatment of cells with the reverse transcriptase inhibitor, phosphonoformic acid (PFA) (26). We included rad50⌬ mutants, which also exhibit a posttranslational increase in transposition (5), in our analysis because of their intermediate transposition phenotype.…”

Novel Function of Rad27 (FEN-1) in Restricting Short-Sequence Recombination

“…Similar to recombination in the rad3, ssl1, and ssl2 mutants described previously (2,3,29,34), SSR was stimulated severalfold in the rad27-null mutant, while recombination between long sequences was unaffected.…”

“…The persistence of these sequences increases the likelihood that they will recombine with other sequences in the genome. Interestingly, these factors also influence Ty1 retrotransposition, which gives rise to distinct genome rearrangements (28,29).…”

“…Recent discoveries suggest a genetic basis for the limitation of short-sequence recombination (SSR) in yeast. Novel mutations in the RAD3, SSL1, and SSL2 genes, which encode components of the nucleotide excision repair (NER) apparatus and transcription factor TFIIH (14,69), disrupt the control of SSR and increase genome rearrangement (2,3,29,34). Genome instability may be due to slow removal of sequences at the ends of broken DNA molecules by an unidentified nuclease or nucleases.…”