Yeast EndoG prevents genome instability by degrading extranuclear DNA species
In metazoans mitochondrial DNA (mtDNA) or retrotransposon cDNA released to cytoplasm are degraded by nucleases to prevent sterile inflammation. It remains unknown whether degradation of these DNA also prevents nuclear genome instability. We used an amplicon sequencing-based method in yeast enabling analysis of millions of DSB repair products. In non-dividing stationary phase cells, Pol4-mediated non-homologous end-joining increases, resulting in frequent insertions of 1-3 nucleotides, and insertions of mtDNA (NUMTs) or retrotransposon cDNA.  Yeast EndoG (Nuc1) nuclease limits insertion of cDNA and transfer of very long mtDNA (>10 kb) to the nucleus, where it forms unstable circles, while promoting the formation of short NUMTs (~45-200 bp). Nuc1 also regulates transfer of extranuclear DNA to nucleus in aging or meiosis. We propose that Nuc1 preserves genome stability by degrading retrotransposon cDNA and long mtDNA, while short NUMTs originate from incompletely degraded mtDNA. This work suggests that nucleases eliminating extranuclear DNA preserve genome stability. Overall design: To study NUMT formation in yeast, we developed a high-throughput amplicon sequencing based method called Break-Ins (Break Insertions) which allows screening of hundreds of thousands of NHEJ products simultaneously for the events carrying NUMTs . We used yeast cells carrying a galactose-inducible HO endonuclease that generates a single double-strand break (DSB) per genome at the MATa locus. This DSB can only be repaired by NHEJ; only the cells that repaired the break imprecisely, altering the HO recognition site, can survive continuous HO induction and form colonies.Cells from an overnight saturated YEPD culture were washed twice with YEP-Raffinose, inoculated into 10 ml YEP-Raffinose and incubated overnight at 30 °C. When the density of the culture was ~2×107 cells/ml, ~1×107 cells were spread on a YEP-galactose 150mm plate and incubated at 30 °C for 5 days. Each culture was spread on 5-7 plates. Some slow growing strains were incubated 1-3 more days. Colony number was counted. Colony numbers were estimated by counting a representative sector of one plate per genotype and multiplying to cover all plates. Cells were collected using a spreader after adding 5 ml water onto each plate. Cells were pooled and mixed vigorously. ~80 µl cells were spun down and the genomic DNA was extracted using standard glass beads and phenol chloroform. The genomic DNA was treated with RNase A overnight. The genomic DNA was dissolved with water and adjusted to a concentration of 10 ng/µl. The construction of the sequencing library was adapted from Illumina's amplicon sequencing protocol #15044223 Rev. B. Two rounds of PCR were performed to construct the sequencing library. For each sample, 3.2 µl genomic DNA, 0.5 µl 10 µM primers and 12.5 µl KAPA HiFi HotStart ReadyMix (Roche, 7958927001) was used for the first round of PCR for a total volume of 25 µl. The forward primers are 66-mers that contain (5' to 3'): 33 bases Illumina adapter sequence (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG), 3 bases unique home index, and 30 bases (GCATAGTCGGGTTTTTCTTTTAGTTTCAGC) targeting the Mata locus which is located 12-41 bp upstream the HO cleavage site. The reverse primers are 66-mers that contain (5' to 3'): 34 bases Illumina adapter sequence (GTCTCGTGGGCTCGGAGATGTG TATAAGAGACAG), 3 bases unique home index, and 29 bases (CAACCACTCTACAAAAC CAAAACCAGGGT) targeting the MATa locus which is located 15-43 bp downstream the HO cleavage site. The following conditions were used for the first round of PCR: 95 °C for 5 min; 22 cycles of 98 °C for 20 s, 65 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. 18µl PCR product was purified with 20µl AMPure XP beads (Beckman Coulter, A63880) and eluted with 52.5 µl 10 mM Tris pH 8.5. For the second round of PCR, 5 µl purified PCR product, 5 µl Nextera XT V2 Index (Illumina, FC-131-2001) primer N7xx, 5 µl index primer S5xx, and 25 µl KAPA HiFi HotStart ReadyMix were used. The total volume of PCR is 50 µl. The following conditions were used for PCR: 95 °C for 5 min; 8 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 3 min; 72 °C for 10 min. 40 µl PCR product was purified with 48 µl AMPure XP beads and eluted with 27.5 µl 10 mM Tris pH 8.5. The DNA concentration of each sample was determined with Qubit dsDNA BR Assay Kit (ThermoFisher, Q32850) and the average size of the DNA was determined with TapeStation. The DNA was diluted to 4 nM using 10 mM Tris pH 8.5. An equal amount of DNA was pooled from ~20 samples into the library. The pooled library and PhiX library (Illumina, FC-110-3001) were denatured with 0.2 M NaOH separately and diluted with pre-chilled HT1 to 12 pM. 540µl pooled library and 60 µl PhiX were mixed, incubated at 96 °C for 2 min and immediately placed in an ice-water bath for 5 min. The denatured combined library was loaded into the MiSeq Reagent Kit v3 (600 cycle) (Illumina, MS-102-3003). The cluster density was ~1100 K/mm2.