RPA and Rad27 limit templated and inverted insertions at DNA breaks
Formation of templated insertions at DNA double-strand breaks (DSBs) is very common in cancer cells. The mechanisms and enzymes regulating these events are largely unknown. Here, we investigated templated insertions in yeast at DSBs using amplicon sequencing across a repaired locus. We document very short (most ~5-34 bp), templated inverted duplications at DSBs. They are generated through a foldback mechanism that utilizes microhomologies adjacent to the DSB. Enzymatic requirements suggest a hybrid mechanism wherein one end requires Pold-mediated synthesis while the other end is captured by nonhomologous end joining (NHEJ) or rarely by alternative end joining (Alt-EJ). This process is exacerbated in mutants with low levels or mutated RPA (rtt105?; rfa1-t33) or extensive resection mutant (sgs1? exo1?). Templated insertions from various distant genomic locations also increase in these mutants as well as in rad27? and originate from fragile regions of the genome. Among complex insertions, common events are insertions of two sequences, originating from the same locus and with inverted orientation. We propose that these inversions are also formed by microhomology-mediated template switching. Taken together, we propose that a shortage of RPA typical in cancer cells is one possible factor stimulating the formation of templated insertions. Overall design: To study insertion at DSBs in yeast, we used a high-throughput amplicon sequencing based method called Break-Ins (Break Insertions) which allows screening of hundreds of thousands of NHEJ products simultaneously. 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't be repaired by homologous recombination; only the cells that repaired the break imprecisely, altering the HO recognition site, can survive continuous HO induction and form colonies.Yeast cells were grown overnight in YPD (1% yeast extract, 2% peptone, 2% dextrose), washed twice with YP-raffinose (1% yeast extract, 2% peptone, 2% raffinose) media, inoculated into 10 mL YP-raffinose media, and incubated at 30 °C overnight. When the cell density was about 2x107/mL, 500 µL cells were spread onto YP-galactose (1% yeast extract, 2% peptone, 2% galactose, 2.5% agar) plates to induce HO expression and the DSB at MATa locus, and incubated at 30 °C for 5 days. Each sample was spread on 5-7 150x15 mm YP-galactose plates. The number of colonies was counted. All colonies on YP-galactose plates were collected, combined, and mixed vigorously. Approximately 80 µL cells were spun down for isolating genomic DNA. Standard glass beads-phenol/chloroform/isoamyl alcohol (25:24:1) method. The DNA was dissolved in water and adjusted to a concentration of 10 ng/µL. The construction of the amplicon sequencing library was adapted from Illumina's protocol #15044223 Rev. B. Briefly, the sequencing libraries were constructed with two rounds of PCR using KAPA HiFi HotStart ReadyMix polymerase (Roche, 7958927001). For the first round of PCR, 3.2 µL genomic DNA and 0.5 µL 10 µM primers were used for a total volume of 25 µL. The forward and reverse primers targeting the MATa locus are located 11 bp upstream or 18 bp downstream of the HO cleavage site respectively, containing Illumina adapter sequence and 3 bases unique home index as described (7). The thermocycling conditions were: 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. After the first round of PCR, 18µL PCR product was purified with an equal volume of AMPure XP beads (Beckman Coulter, A63880). The DNA was eluted with 52.5 µL of 10 mM Tris (pH 8.5) and used as the templates for the second round of PCR. For the second round of PCR, 5 µl template DNA, 5 µL index primer N7xx and 5 µL index primer S5xx from Nextera XT V2 Index kit (Illumina, FC-131-2001) were used for the total volume of 50 µL. The following conditions were used for the second round of 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. Then, the PCR product was purified with 1.2x volume of AMPure XP beads and eluted with 10 mM Tris pH 8.5. The average size of each sample was measured with TapeStation and the DNA concentration was measured with Qubit dsDNA BR Assay Kit (ThermoFisher, Q32850). Then, the DNA was diluted to a concentration of 4 nM. An equal volume of DNA from ~20 samples was pooled into the library. The 0.2 M NaOH was used to denature the pooled library and PhiX library (Illumina, FC-110-3001). After diluting the libraries with pre-chilled HT1 to 12 pM, pooled library and PhiX library were mixed with the ratio 9:1, further denatured by incubating at 96 °C for 2 min and immediately put in an ice-water bath for 5 min. The MiSeq and Reagent Kit v3 (600 cycles) (Illumina, MS-102-3003) were used for sequencing. The cluster density for all libraries was ~1100 K/mm2.