Assessing contributions of DNA sequences at the 3' end of a yeast gene on yFACT, RNA polymerase II, and nucleosome occupancy.

OBJECTIVE: In past work in budding yeast, we identified a nucleosomal region required for proper interactions between the histone chaperone complex yFACT and transcribed genes. Specific histone mutations within this region cause a shift in yFACT occupancy towards the 3' end of genes, a defect that we have attributed to impaired yFACT dissociation from DNA following transcription. In this work we wished to assess the contributions of DNA sequences at the 3' end of genes in promoting yFACT dissociation upon transcription termination. RESULTS: We generated fourteen different alleles of the constitutively expressed yeast gene PMA1, each lacking a distinct DNA fragment across its 3' end, and assessed their effects on occupancy of the yFACT component Spt16. Whereas most of these alleles conferred no defects on Spt16 occupancy, one did cause a modest increase in Spt16 binding at the gene's 3' end. Interestingly, the same allele also caused minor retention of RNA Polymerase II (Pol II) and altered nucleosome occupancy across the same region of the gene. These results suggest that specific DNA sequences at the 3' ends of genes can play roles in promoting efficient yFACT and Pol II dissociation from genes and can also contribute to proper chromatin architecture.

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ-dependent mechanism.

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease activity. Foldback priming at DNA double-strand breaks (DSBs) is one mechanism proposed for the generation of inverted duplications. However, the other pathway steps have not been fully elucidated. Here, we show that a DSB induced near natural inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity, but not Rad1 nuclease, trims the heterologous flaps formed after foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric inversion chromosome. Finally, we show that stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences or by deletion of one centromere.

Long-range DNA end resection supports homologous recombination by checkpoint activation rather than extensive homology generation.

Homologous recombination (HR), the high-fidelity mechanism for double-strand break (DSB) repair, relies on DNA end resection by nucleolytic degradation of the 5'-terminated ends. However, the role of long-range resection mediated by Exo1 and/or Sgs1-Dna2 in HR is not fully understood. Here, we show that Exo1 and Sgs1 are dispensable for recombination between closely linked repeats, but are required for interchromosomal repeat recombination in Saccharomyces cerevisiae. This context-specific requirement for long-range end resection is connected to its role in activating the DNA damage checkpoint. Consistent with this role, checkpoint mutants also show a defect specifically in interchromosomal recombination. Furthermore, artificial activation of the checkpoint partially restores interchromosomal recombination to exo1∆ sgs1∆ cells. However, cell cycle delay is insufficient to rescue the interchromosomal recombination defect of exo1∆ sgs1∆ cells, suggesting an additional role for the checkpoint. Given that the checkpoint is necessary for DNA damage-induced chromosome mobility, we propose that the importance of the checkpoint, and therefore long-range resection, in interchromosomal recombination is due to a need to increase chromosome mobility to facilitate pairing of distant sites. The need for long-range resection is circumvented when the DSB and its repair template are in close proximity.

Double-strand breaks induce inverted duplication chromosome rearrangements by a DNA polymerase δ and Rad51-dependent mechanism.

Inverted duplications, also known as foldback inversions, are commonly observed in cancers and are the major class of chromosome rearrangement recovered from yeast cells lacking Mre11 nuclease. Foldback priming at naturally occurring inverted repeats is one mechanism proposed for the generation of inverted duplications. However, the initiating lesion for these events and the mechanism by which they form has not been fully elucidated. Here, we show that a DNA double-strand break (DSB) induced near natural short, inverted repeats drives high frequency inverted duplication in Sae2 and Mre11-deficient cells. We find that DNA polymerase δ proof-reading activity acts non-redundantly with Rad1 nuclease to remove heterologous tails formed during foldback annealing. Additionally, Pol32 is required for the generation of inverted duplications, suggesting that Pol δ catalyzes fill-in synthesis primed from the foldback to create a hairpin-capped chromosome that is subsequently replicated to form a dicentric isochromosome. Stabilization of the dicentric chromosome after breakage involves telomere capture by non-reciprocal translocation mediated by repeat sequences and requires Rad51.