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Timely repair of chromosomal double strand breaks is required for genome integrity and cellular viability. The Polymerase Theta-mediated End Joining pathway has an important role in resolving these breaks and is essential in cancers defective in other DNA repair pathways, thus is an emerging therapeutic target. It requires annealing of 2-6 nucleotides of complementary sequence – microhomologies – that are adjacent to the broken ends, followed by initiation of end-bridging DNA synthesis by Polymerase theta. However, the other pathway steps remain inadequately defined, and the enzymes required for them are unknown. Here we demonstrate additional requirements for exonucleolytic digestion of unpaired 3’ tails before Polymerase theta can initiate synthesis, then a switch to a more accurate, processive, and strand-displacing polymerase to complete repair. We show the replicative polymerase, Polymerase delta, is required for both steps; its 3’ to 5’ exonuclease activity for flap trimming, then its polymerase activity for extension and completion of repair. The enzymatic steps that are essential and specific to this pathway are mediated by two separate, sequential engagements of the two polymerases. We show the requisite coupling of these steps together is facilitated by physical association of the two polymerases. This pairing of Polymerase Delta with a polymerase capable of end-bridging synthesis, Polymerase theta, may explain why the normally high-fidelity Polymerase delta participates in genome de-stabilizing processes like mitotic DNA synthesis and microhomology-mediated break induced replication.

Significance Repair of chromosome breaks by polymerase theta-mediated end joining (TMEJ) requires short sequence identities in flanking DNA (microhomologies)—a sequence-context constraint that is unique among DNA repair pathways. Though microhomologies have a central role in TMEJ, it has been uncertain whether an organized mechanism to identify them even exists. Using a combination of chromosomal and extrachromosomal substrates, we describe how polymerase theta efficiently locates microhomologies when present, and creates them de novo when absent. We show how this generates a pattern of microhomology-mediated end joining products that is sufficiently distinct from other end joining pathways and that it can be used as a biomarker for TMEJ activity in cancer genomes., 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.

The DNA polymerase theta (Polθ)-dependent end joining (TMEJ) pathway for repair of chromosomal double strand breaks (DSBs) is essential in cells deficient in other DSB repair pathways, including hereditary breast cancers defective in homologous recombination. Strand-break activated poly(ADP) ribose polymerase 1 (PARP1) has been implicated in TMEJ, but the modest specificity of existing TMEJ assays means the extent of effect and the mechanism behind it remain unclear. We describe here a series of TMEJ assays with improved specificity and show ablation of PARP activity reduces TMEJ activity 2-4-fold. The reduction in TMEJ is attributable to a reduction in the 5’ to 3’ resection of DSB ends that is essential for engagement of this pathway and is compensated by increased repair by the nonhomologous-end joining pathway. This limited role for PARP activity in TMEJ helps better rationalize the combined employment of inhibitors of PARP and Polθ in cancer therapy.

Many cancer therapies, including radiotherapy, induce DSBs as the major driving mechanism for inducing cancer cell death. Thus, modulating DSB repair has immense potential for radiosensitization, although such interventions must be carefully designed to be tumor selective to ensure that normal tissue toxicities are not also increased. Here, we review mechanisms of error-prone DSB repair through a highly efficient process called end joining. There are two major pathways of end-joining repair: non-homologous end joining (NHEJ) and alternative end joining (a-EJ), both of which can be selectively upregulated in cancer and thus represent attractive therapeutic targets for radiosensitization. These EJ pathways each have therapeutically targetable pioneer factors - DNA-dependent protein kinase catalytic subunit (DNA-PKcs) for NHEJ and DNA Polymerase Theta (Pol θ) for a-EJ. We summarize the current status of therapeutic targeting of NHEJ and a-EJ to enhance the effects of radiotherapy - focusing on challenges that must be overcome and opportunities that require further exploration. By leveraging preclinical insights into mechanisms of altered DSB repair programs in cancer, selective radiosensitization through NHEJ and/or a-EJ targeting remains a highly attractive avenue for ongoing and future clinical investigation.

Genome integrity and genome engineering require efficient repair of DNA double-strand breaks (DSBs) by non-homologous end joining (NHEJ), homologous recombination (HR), or alternative end-joining pathways. Here we describe two complementary methods for marker-free quantification of DSB repair pathway utilization at Cas9-targeted chromosomal DSBs in mammalian cells. The first assay features the analysis of amplicon next-generation sequencing data using ScarMapper, an iterative break-associated alignment algorithm to classify individual repair products based on deletion size, microhomology usage, and insertions. The second assay uses repair pathway-specific droplet digital PCR assays (‘PathSig-dPCR’) for absolute quantification of signature DSB repair outcomes. We show that ScarMapper and PathSig-dPCR enable comprehensive assessment of repair pathway utilization in different cell models, after a variety of experimental perturbations. We use these assays to measure the differential impact of DNA end resection on NHEJ, HR and polymerase theta-mediated end joining (TMEJ) repair. These approaches are adaptable to any cellular model system and genomic locus where Cas9-mediated targeting is feasible. Thus, ScarMapper and PathSig-dPCR allow for systematic fate mapping of a targeted DSB with facile and accurate quantification of DSB repair pathway choice at endogenous chromosomal loci.

TP53 deficiency in cancer is associated with poor patient outcomes and resistance to DNA damaging therapies. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Using live cell imaging of DNA double-strand breaks (DSBs) and cell cycle state transitions, we show that p53-deficient cells exhibit accelerated repair of radiomimetic-induced DSBs arising in S phase. Low-dose DNA-dependent protein kinase (DNA-PK) inhibition increases the S-phase DSB burden in p53-deficient cells, resulting in elevated rates of mitotic catastrophe. However, a subset of p53-deficient cells exhibits intrinsic resistance to radiomimetic-induced DSBs despite DNA-PK inhibition. We show that p53-deficient cells under DNA-PK inhibition utilize DNA polymerase theta (Pol θ)-mediated end joining repair to promote their viability in response to therapy-induced DSBs. Pol θ inhibition selectively increases S-phase DSB burden after radiomimetic therapy and promotes prolonged G2 arrest. Dual inhibition of DNA-PK and Pol θ restores radiation sensitivity in p53-deficient cells as well as in p53-mutant breast cancer cell lines. Thus, combination targeting of DNA-PK- and Pol θ-dependent end joining repair represents a promising strategy for overcoming resistance to DNA damaging therapies in p53-deficient cancers.

DNA repair deficiencies have become an increasingly promising target for novel therapeutics within the realm of clinical oncology. Recently, several inhibitors of Poly(ADP-ribose) Polymerases (PARPs) have received approval for the treatment of cancers primarily with deleterious mutations in the homologous recombination (HR) proteins, BRCA1 and BRCA2. Despite numerous clinical trials which have been completed or are currently ongoing, the mechanism of action by which PARP inhibitors selectively kill tumor cells is poorly understood. While many believe “trapping” of PARP proteins to DNA at sites of damage is the most important determinant driving cytotoxicity by these drugs, clinically effective inhibitors exist with a diverse range of PARP-trapping qualities. These findings suggest that characterization of inhibitors as strong versus weak trappers does not properly capture the intra-class characteristics of these drugs. Here, we use a novel, targeted DNA damage response (DDR) CRISPR/Cas9 screening library to reveal heterogenous genetic dependencies on the base excision repair (BER) pathway for PARP inhibitors, which is not correlated with PARP trapping ability or catalytic inhibition of PARP. These findings demonstrate that inhibition of PARylation and induction of PARP trapping are not the only factors contributing to distinct biological activity for different PARP inhibitors, and they provide insight into the optimal choice of PARP inhibitors for use in the setting of specific DDR defects.AUTHOR SUMMARYTargeted cancer therapies rely on our general understanding of which genetic mutations are involved in both sensitivity and resistance to such anticancer agents. In this study, we describe the use of functional genetic screening to evaluate the role of various DNA repair proteins in response to inhibitors of PARP, a quintessential example of targeted therapy. While PARP inhibitors are best known for their utility in cancers with homologous recombination defects, we show that some inhibitors within this class may have additional functionality in cancers with deficient base excision repair. These findings highlight not only the importance of PARP inhibitor selection in the appropriate context, but also the mechanistic differences that exist within this class of inhibitors. It is our hope that our findings will inspire future work evaluating the use of specific PARP inhibitor selection in designing clinical trials to further expand the use of PARP inhibitors beyond tumors with homologous recombination deficiencies.

TP53 mutations in cancer are associated with poor patient outcomes and resistance to DNA damaging therapies1–3. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Here, we show that p53-deficient cells exhibit hyperactive repair of therapy-induced DNA double strand breaks (DSBs), which is suppressed by inhibition of DNA-dependent protein kinase (DNA-PK). Single-cell analyses of DSB repair kinetics and cell cycle state transitions reveal an essential role for DNA-PK in suppressing S phase DNA damage and mitotic catastrophe in p53-deficient cells. Yet, a subset of p53-deficient cells exhibit intrinsic resistance to therapeutic DSBs due to a repair pathway that is not sensitive to DNA-PK inhibition. We show that p53 deficiency induces overexpression of DNA Polymerase Theta (Pol θ), which mediates an alternative end-joining repair pathway that becomes hyperactivated by DNA-PK inhibition4. Combined inhibition of DNA-PK and Pol θ restores therapeutic DNA damage sensitivity in p53-deficient cells. Thus, our study identifies two targetable DSB end joining pathways that can be suppressed as a strategy to overcome resistance to DNA-damaging therapies in p53-deficient cancers.

DNA polymerase theta (Pol θ)-mediated end joining (TMEJ) has been implicated in the repair of chromosome breaks, but its cellular mechanism and role relative to canonical repair pathways are poorly understood. We show that it accounts for most repairs associated with microhomologies and is made efficient by coupling a microhomology search to removal of non-homologous tails and microhomology-primed synthesis across broken ends. In contrast to non-homologous 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 to sustain cell viability and genome stability by rescuing chromosome break repair when resection is misregulated or NHEJ is compromised.

BRCA1 promotes the DNA end-resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, or if mutant proteins retaining partial activity can complement one another, is unclear, and could impact the severity of BRCA1-associated Fanconi anemia (FA). Here, we generated a Brca1(CC) mouse with a coiled-coil (CC)-domain deletion. Brca1(CC/CC) mice are born at low frequencies and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1(Δ11), which is homozygous lethal, generated Brca1(CC/Δ11) mice at Mendelian frequencies that were indistinguishable from Brca1(+/+) mice. Brca1(CC) and Brca1(Δ11) proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1(CC) and Brca1(Δ11) alleles represent separation-of-function mutations, which combine to provide a level of HR that is sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.

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 Brca1ΔC/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. : Using a Brca1ΔC mouse model and a panel of BRCA1 mutant cancer cell lines, Nacson et al. show that 53BP1 loss of function induced homologous recombination and PARP inhibitor resistance is suboptimal in the absence of hypomorphic BRCA1 proteins that retain the coiled-coil domain and ability to interact with PALB2. Keywords: BRCA1, 53BP1, homologous recombination, PARP inhibitors, resistance

The Fanconi anemia protein PALB2, also known as FANCN, protects genome integrity by regulating DNA repair and cell cycle checkpoints. Exactly how PALB2 functions may be temporally coupled with detection and signaling of DNA damage is not known. Intriguingly, we found that PALB2 is transformed into a hyperphosphorylated state in response to ionizing radiation (IR). IR treatment specifically triggered PALB2 phosphorylation at Ser-157 and Ser-376 in manners that required the master DNA damage response kinase Ataxia telangiectasia mutated, revealing potential mechanistic links between PALB2 and the Ataxia telangiectasia mutated-dependent DNA damage responses. Consistently, dysregulated PALB2 phosphorylation resulted in sustained activation of DDRs. Full-blown PALB2 phosphorylation also required the breast and ovarian susceptible gene product BRCA1, highlighting important roles of the BRCA1-PALB2 interaction in orchestrating cellular responses to genotoxic stress. In summary, our phosphorylation analysis of tumor suppressor protein PALB2 uncovers new layers of regulatory mechanisms in the maintenance of genome stability and tumor suppression.

Under most microscopes, the fluorescent signals emitted from biological structures, such as vesicles, as well as the signals from single molecules will appear as small puncta, which contribute to a Gaussian-like distribution. Accurate segmentation of these spots will fundamentally affect our interpretation of a specific biological progress. Because of the complicated backgrounds in images, many algorithms fail to identify all of the interesting signals; the tremendous amount of time required for algorithms to process large datasets can also decrease their utility. Here, we introduce an excellent robust detection method based on the machine learning algorithm AdaBoost, which outperforms threshold-based segmentation, wavelets, and FDA under most situations. We also provide a GPU/multi-core CPU implementation of this algorithm; this implementation accelerates the algorithm approximately 10- and 7-fold acceleration compared with a single CPU implementation. The great reduction of time should make this method a promising candidate in the processing of large datasets. Furthermore, we demonstrate the use of our algorithm on true fluorescent micrographs, and the results show that machine learning-based detection methods outperform the four other previously reported methods.

Ubiquitin signals emanating from DNA double-strand breaks (DSBs) trigger the ordered assembly of DNA damage mediator and repair proteins. This highly orchestrated process is accomplished, in part, through the concerted action of the RNF8 and RNF168 E3 ligases, which have emerged as core signaling intermediates that promote DSB-associated ubiquitylation events. In this study, we report the identification of RNF169 as a negative regulator of the DNA damage signaling cascade. We found that RNF169 interacted with ubiquitin structures and relocalized to DSBs in an RNF8/RNF168-dependent manner. Moreover, ectopic expression of RNF169 attenuated ubiquitin signaling and compromised 53BP1 accumulation at DNA damage sites, suggesting that RNF169 antagonizes RNF168 functions at DSBs. Our study unveils RNF169 as a component in DNA damage signal transduction and adds to the complexity of regulatory ubiquitylation in genome stability maintenance., link_to_OA_fulltext

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.

An ultrasensitive and highly specific electrochemical aptasensor for detection of thrombin based on gold nanoparticles and thiocyanuric acid is presented. For this proposed aptasensor, aptamerI was immobilized on the magnetic nanoparticles, aptamerII was labeled with gold nanoparticles. The magnetic nanoparticle was used for separation and collection, and gold nanoparticle offered excellent electrochemical signal transduction. Through the specific recognition for thrombin, a sandwich format of magnetic nanoparticle/thrombin/gold nanoparticle was fabricated, and the signal amplification was further implemented by forming network-like thiocyanuric acid/gold nanoparticles. A significant sensitivity enhancement had been obtained, and the detection limit was down to 7.82 aM. The presence of other proteins such as BSA and lysozyme did not affect the detection of thrombin, which indicates a high specificity of thrombin detection could be achieved. This electrochemical aptasensor is expected to have wide applications in protein monitoring and disease diagnosis.

The molecular mechanisms by which dense core vesicles (DCVs) translocate, tether, dock and prime are poorly understood. In this study, Caenorhabditis elegans was used as a model organism to study the function of Rab proteins and their effectors in DCV exocytosis. RAB-27/AEX-6, but not RAB-3, was found to be required for peptide release from neurons. By analyzing the movement of DCVs approaching the plasma membrane using total internal reflection fluorescence microscopy, we demonstrated that RAB-27/AEX-6 is involved in the tethering of DCVs and that its effector rabphilin/RBF-1 is required for the initial tethering and subsequent stabilization by docking.