DNA polymerase theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3′ single-stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3′ terminus and favoring bidirectional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3 to 30 bp of sequence that is identical to flanking DNA (“templated” insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification and shows how it both mitigates limitations implicit in the microhomology requirement and generates distinctive genomic scars associated with pathogenic genome instability.

19DNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs 20 chromosome breaks. It requires resection of broken ends to generate long, 3' 21 single stranded DNA tails, annealing of complementary sequence segments 22 (microhomologies) in these tails, followed by microhomology-primed synthesis 23 sufficient to resolve broken ends. The means by which microhomologies are 24 identified is thus a critical step in this pathway, but is not understood. Here we 25 show microhomologies are identified by a scanning mechanism initiated from the 26 3' terminus and favoring bi-directional progression into flanking DNA, typically to a 27 maximum of 15 nucleotides into each flank. Polymerase theta is frequently 28 insufficiently processive to complete repair of breaks in microhomology-poor, AT-29 rich regions. Aborted synthesis leads to one or more additional rounds of 30 microhomology search, annealing, and synthesis; this promotes complete repair 31 in part because earlier rounds of synthesis generate microhomologies de novo that 32 are sufficiently long that synthesis is more processive. Aborted rounds of synthesis 33 are evident in characteristic genomic scars as insertions of 3-30 bp of sequence 34 that is identical to flanking DNA ("templated" insertions). Templated insertions are 35 present at higher levels in breast cancer genomes from patients with germline 36 BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our 37 work thus describes the mechanism for microhomology identification, and shows 38 both how it mitigates limitations implicit in the microhomology requirement, and 39 3 generates distinctive genomic scars associated with pathogenic genome 40 instability. 41 42 99 100 Materials and Methods 101 Cell lines 102 Polq -/-Mouse Embryonic Fibroblasts (MEFs) were generated and immortalized 103 with T antigen as described (Yousefzadeh et al., 2014) from Polq-null mice 104 generated by conventional knock-out (Shima, Munroe and Schimenti, 2004) that 105 were obtained from Jackson Laboratories and maintained on a C57BL/6J 106 background. Pol θ was expressed by introducing wt POLQ human cDNA by 107 6 lentiviral infection, with cells maintained in medium containing 4 μg/ml of 108 puromycin. Cells were incubated at 37 °C, 5% CO2 and cultured in DMEM (Gibco) 109 with 10% Fetal Bovine Serum (VWR Life Science Seradigm) and Penicillin (5 U/ml, 110Sigma). These lines and variants described below were confirmed to be free of 111 mycoplasma contamination by a qPCR (Janetzko et al., 2014) with a detection limit 112 below 10 genomes/1ml. Cell lines were additionally selected at random for third 113 party validation of PCR results using Hoechst staining (Battaglia et al., 1994). 114 Extrachromosomal assay 115 As has been described previously (Wyatt et al., 2016), extrachromosomal 116 substrates consist of a 557 bp core DNA duplex ligated to head and tail caps with 117 end structures that were varied as described in each figure. 75 ng of these 118 substrates were electroporated into 200,000 cel...