Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.

OBJECTIVE: The PARP inhibitor (PARPi), Talazoparib (BMN673), effectively and specifically radiosensitizes cancer cells. Radiosensitization is mediated by a shift in the repair of ionizing radiation (IR)-induced DNA double-strand breaks (DSBs) toward PARP1-independent, alternative end-joining (alt-EJ). DNA polymerase theta (Polθ) is a key component of this PARP1-independent alt-EJ pathway and we show here that its inhibition can further radiosensitize talazoparib-treated cells. The purpose of the present work is to explore mechanisms and dynamics underpinning enhanced talazoparib radiosensitization by Polθ inhibitors in HR-proficient cancer cells.
METHODS: Radiosensitization to PARPis, talazoparib, olaparib, rucaparib and veliparib was assessed by clonogenic survival. Polθ-proficient and -deficient cells were treated with PARPis and/or with the Polθ inhibitors ART558 or novobiocin. The role of DNA end-resection was studied by down-regulating CtIP and MRE11 expression using siRNAs. DSB repair was assessed by scoring γH2AX foci. The formation of chromosomal abnormalities was assessed as evidence of alt-EJ function using G2-specific cytogenetic analysis.
RESULTS: Talazoparib exerted pronounced radiosensitization that varied among the tested cancer cell lines; however, radiosensitization was undetectable in normal cells. Other commonly used PARPis, olaparib, veliparib, or rucaparib were ineffective radiosensitizers under our experimental conditions. Although genetic ablation or pharmacological inhibition of Polθ only mildly radiosensitized cancer cells, talazoparib-treated cells were markedly further radiosensitized. Mechanistically, talazoparib shunted DSBs to Polθ-dependent alt-EJ by enhancing DNA end-resection in a CtIP- and MRE11-dependent manner - an effect detectable at low, but not high IR doses. Chromosomal translocation analysis in talazoparib-treated cells exposed to Polθ inhibitors suggested that PARP1- and Polθ-dependent alt-EJ pathways may complement, but also back up each other.
CONCLUSIONS: We propose that talazoparib promotes low-dose, CtIP/MRE11-dependent resection and increases the reliance of irradiated HR-proficient cancer cells, on Polθ-mediated alt-EJ. The combination of Polθ inhibitors with talazoparib suppresses this option and causes further radiosensitization. The results suggest that Polθ inhibition may be exploited to maximize talazoparib radiosensitization of HR-proficient tumors in the clinic.

DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.

OBJECTIVE: BRCA1/2 mutations in breast cancer cells impair homologous recombination and promote alternative end joining (Alt-EJ) for DNA-damage repair. DNA polymerase theta, encoded by POLQ, plays a crucial role in Alt-EJ, making it a potential therapeutic target, particularly in BRCA1/2-mutant cancers. Methionine restriction is a promising approach to target cancer cells due to their addiction to this amino acid. The present study investigated the expression of POLQ in BRCA1/2 wild-type and BRCA1-mutant breast cancer cells under methionine restriction.
METHODS: POLQ mRNA expression was measured using qRT-PCR in BRCA1/2 wild-type (MDA-MB-231) and BRCA1- mutant (HCC1937 and MDA-MB-436) breast-cancer cells under normal, or serum-restricted, or serum- and methionine-restricted conditions.
RESULTS: Compared to BRCA1/2 wild-type cells, BRCA1-mutant cells displayed significantly higher basal POLQ expression in normal medium. Methionine restriction further increased POLQ expression in the BRCA1-mutant cells but decreased it in the BRCA1/2 wild-type cells.
CONCLUSIONS: The present findings suggest that methionine restriction showed differential effects on POLQ expression, potentially impacting Alt-EJ activity, in BRCA1/2 wild-type and BRCA1-mutant breast-cancer cells. Further investigation is needed to explore the potential of combining methionine restriction with DNA-repair inhibitors, such as PARP inhibitors, to overcome drug resistance in BRCA1/2 mutant cancers.

Theta-mediated end joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend ssDNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both ssDNA and RNA substrates by unimolecular stem-loop synthesis initiated by only two 3\' terminal base pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem-loop synthesis competes with bimolecular end joining, even when a longer terminal microhomology for end joining is available. Both reactions are partially suppressed by the ssDNA-binding protein replication protein A. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare among human DNA polymerases, but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem-loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.

According to the CDC, both Pfizer and Moderna COVID-19 vaccines contain nucleoside-modified messenger RNA (mRNA) encoding the viral spike glycoprotein of severe acute respiratory syndrome caused by corona virus (SARS-CoV-2), administered via intramuscular injections. Despite their worldwide use, very little is known about how nucleoside modifications in mRNA sequences affect their breakdown, transcription and protein synthesis. It was hoped that resident and circulating immune cells attracted to the injection site make copies of the spike protein while the injected mRNA degrades within a few days. It was also originally estimated that recombinant spike proteins generated by mRNA vaccines would persist in the body for a few weeks. In reality, clinical studies now report that modified SARS-CoV-2 mRNA routinely persist up to a month from injection and can be detected in cardiac and skeletal muscle at sites of inflammation and fibrosis, while the recombinant spike protein may persist a little over half a year in blood. Vaccination with 1-methylΨ (pseudouridine enriched) mRNA can elicit cellular immunity to peptide antigens produced by +1 ribosomal frameshifting in major histocompatibility complex-diverse people. The translation of 1-methylΨ mRNA using liquid chromatography tandem mass spectrometry identified nine peptides derived from the mRNA +1 frame. These products impact on off-target host T cell immunity that include increased production of new B cell antigens with far reaching clinical consequences. As an example, a highly significant increase in heart muscle 18-flourodeoxyglucose uptake was detected in vaccinated patients up to half a year (180 days). This review article focuses on medical biochemistry, proteomics and deutenomics principles that explain the persisting spike phenomenon in circulation with organ-related functional damage even in asymptomatic individuals. Proline and hydroxyproline residues emerge as prominent deuterium (heavy hydrogen) binding sites in structural proteins with robust isotopic stability that resists not only enzymatic breakdown, but virtually all (non)-enzymatic cleavage mechanisms known in chemistry.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

DNA double-strand breaks (DSBs) represent one of the most severe threats to genomic integrity, demanding intricate repair mechanisms within eukaryotic cells. A diverse array of factors orchestrates the complex choreography of DSB signaling and repair, encompassing repair pathways, such as non-homologous end-joining, homologous recombination, and polymerase-θ-mediated end-joining. This review looks into the intricate decision-making processes guiding eukaryotic cells towards a particular repair pathway, particularly emphasizing the processing of two-ended DSBs. Furthermore, we elucidate the transformative role of Cas9, a site-specific endonuclease, in revolutionizing our comprehension of DNA DSB repair dynamics. Additionally, we explore the burgeoning potential of Cas9\'s remarkable ability to induce sequence-specific DSBs, offering a promising avenue for precise targeting of tumor cells. Through this comprehensive exploration, we unravel the intricate molecular mechanisms of cellular responses to DSBs, shedding light on both fundamental repair processes and cutting-edge therapeutic strategies.