Summary DNA Polymerase theta (Pol θ) mediated end-joining (TMEJ) has been implicated in repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways is poorly understood. We show it accounts for most repair associated with microhomologies, and is made efficient by coupling a microhomology search to removal of nonhomologous tails and microhomology-primed synthesis across broken ends. In contrast to nonhomologous end-joining (NHEJ), TMEJ efficiently repairs end structures expected after aborted homology-directed repair (5′ to 3′ resected ends) or replication fork collapse. It typically does not compete with canonical repair pathways, but in NHEJ-deficient cells is engaged more frequently and protects against translocation. Cell viability is also severely impaired upon combined deficiency in Pol θ and a factor that antagonizes end resection (Ku or 53BP1). TMEJ thus helps sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.

Polymerase theta (Pol θ, gene name Polq) is a widely conserved DNA polymerase that mediates a microhomology-mediated, error-prone, double strand break (DSB) repair pathway, referred to as Theta Mediated End Joining (TMEJ). Cells with homologous recombination deficiency are reliant on TMEJ for DSB repair. It is unknown whether deficiencies in other components of the DNA damage response (DDR) also result in Pol θ addiction. Here we use a CRISPR genetic screen to uncover 140 Polq synthetic lethal (PolqSL) genes, the majority of which were previously unknown. Functional analyses indicate that Pol θ/TMEJ addiction is associated with increased levels of replication-associated DSBs, regardless of the initial source of damage. We further demonstrate that approximately 30% of TCGA breast cancers have genetic alterations in PolqSL genes and exhibit genomic scars of Pol θ/TMEJ hyperactivity, thereby substantially expanding the subset of human cancers for which Pol θ inhibition represents a promising therapeutic strategy.

Highlights d A Brca1 coiled-coil (CC) mutation results in FA in mice d Brca1 exon 11 coding region is required for DNA end resection d Brca1 CC domain is essential for efficient RAD51 loading d Combining complementary mutant alleles partially restores HR

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.

Background: Ubiquitylation plays important roles in DNA damage signal transduction. However, mechanistic details that drive these ubiquitin-dependent signals at DNA breaks remain to be determined. Results: RNF169 localized at DNA double-strand breaks (DSBs), inhibited DNA damage-induced ubiquitin formation, and attenuated 53BP1 accumulation. Conclusion: RNF169 antagonizes ubiquitin signaling at DNA DSBs. Significance: RNF169 represents a negative regulator of the ubiquitin-dependent DNA damage signaling cascade.

SUMMARY BRCA1 functions in homologous recombination (HR) both up- and downstream of DNA end resection. However, in cells with 53BP1 gene knockout (KO), BRCA1 is dispensable for the initiation of resection, but whether BRCA1 activity is entirely redundant after end resection is unclear. Here, we found that 53bp1 KO rescued the embryonic viability of a Brea1ΔC/ΔC mouse model that harbors a stop codon in the coiled-coil domain. However, Brca1ΔC/ΔC;53bp1−/− mice were susceptible to tumor formation, lacked Rad51 foci, and were sensitive to PARP inhibitor (PARPi) treatment, indicative of suboptimal HR. Furthermore, BRCA1 mutant cancer cell lines were dependent on truncated BRCA1 proteins that retained the ability to interact with PALB2 for 53BP1 KO induced RAD51 foci and PARPi resistance. Our data suggest that the overall efficiency of 53BP1 loss of function induced HR may be BRCA1 mutation dependent. In the setting of 53BP1 KO, hypomorphic BRCA1 proteins are active downstream of end resection, promoting RAD51 loading and PARPi resistance.

PCNA is a central scaffold that coordinately assembles replication and repair machineries at DNA replication forks for faithful genome duplication. Here, we describe TRAIP (RNF206) as a novel PCNA-interacting factor that has important roles during mammalian replicative stress responses. We show that TRAIP encodes a nucleolar protein that migrates to stalled replication forks, and that this is accomplished by its targeting of PCNA via an evolutionarily conserved PIP box on its C terminus. Accordingly, inactivation of TRAIP or its interaction with the PCNA clamp compromised replication fork recovery and progression, and leads to chromosome instability. Together, our findings establish TRAIP as a component of the mammalian replicative stress response network, and implicate the TRAIP-PCNA axis in recovery of stalled replication forks.

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...