Topoisomerase inhibitors are widely used chemotherapeutic drugs. They covalently trap the target topoisomerase on DNA, which interferes with the progression of replication and transcription machinery and induces lethal DNA damage. The ability of cells to repair this damage is a key determinant of the effectiveness of these drugs and elucidation of the repair pathways is of great clinical relevance. In this study, I investigate the role of Sae2 protein in protecting yeast cells from camptothecin, a Topoisomerase I inhibitor. Sae2 is a key component of the DNA double strand break repair pathway and works in collaboration with the Mre11-Rad50-Xrs2 (MRX) protein complex in homologous recombination mediated repair. Sae2 null cells are highly sensitive to camptothecin in survival assays. I show that this sensitivity is dependent on active replication as well as transcription in vivo. Sae2 preferentially localizes to highly transcribed regions after camptothecin exposure in S phase, suggesting that it has a role in repair of TopoI-DNA conjugates at those sites. We find that camptothecin sensitivity in the absence of Sae2 can be rescued by over-expression of Sen1, an RNA:DNA helicase that is part of the transcription termination NRD complex. Sen1 removes R-loops that form in the negatively supercoiled region behind a stalled or slow transcription unit. Sen1 over-expression also rescues a strain containing both a sae2 null allele and an Mre11 nuclease-deficient allele. I show that R-loops accumulate at the highly transcribed rDNA region after camptothecin exposure in the absence of Sae2 function and that Sen1 over-expression reduces this accumulation. I propose that Sae2 and Mre11 have a role in processing the R loops induced by TopoI-DNA conjugates at highly transcribed regions in the genome.
It has been shown that Sae2 is a structure-specific endonuclease with a preference for 5’ flaps and ssDNA/dsDNA junctions in vitro. It also stimulates the nuclease activity of Mre11 on DNA ends containing protein adducts. In this study, I identify two mutant alleles of Sae2 that separate these two biochemical activities. The D285P K288P Sae2 mutant is proficient in the Sae2 nuclease activity but deficient for the stimulation of Mre11 activity on protein-blocked DNA ends. The E161P K163P mutation renders the protein deficient for its nuclease activity but the mutant can stimulate Mre11 activity. The survival complementation assay indicates that stimulation of Mre11 activity is the primary function of Sae2 and that its nuclease activity is required only in the absence of Mre11 nuclease activity. Sae2 is implicated in a variety of roles in DNA double-strand break repair and signaling pathways including stimulating resection, modulating Tel1 activation, removing MRX from the DSB sites, processing hairpin intermediates, removing Spo11 from meiotic DNA breaks and processing Topo1-DNA adducts. The mutant alleles described in this study provide useful reagents to elucidate the role of Sae2 enzymatic activity and its coordination with Mre11 nuclease activity in different biological functions.