Sequential requirements for distinct Polθ domains during theta-mediated end joining.

DNA polymerase θ (Polθ) plays a central role in a DNA double-strand break repair pathway termed theta-mediated end joining (TMEJ). TMEJ functions by pairing short-sequence "microhomologies" (MHs) in single-stranded DNA at each end of a break and subsequently initiating DNA synthesis. It is not known how the Polθ helicase domain (HD) and polymerase domain (PD) operate to bring together MHs and facilitate repair. To resolve these transient processes in real time, we utilized in vitro single-molecule FRET approaches and biochemical analyses. We find that the Polθ-HD mediates the initial capture of two ssDNA strands, bringing them in close proximity. The Polθ-PD binds and stabilizes pre-annealed MHs to form a synaptic complex (SC) and initiate repair synthesis. Individual synthesis reactions show that Polθ is inherently non-processive, accounting for complex mutational patterns during TMEJ. Binding of Polθ-PD to stem-loop-forming sequences can substantially limit synapsis, depending on the available dNTPs and sequence context.

Biomarker-Guided Development of DNA Repair Inhibitors.

Anti-cancer drugs targeting the DNA damage response (DDR) exploit genetic or functional defects in this pathway through synthetic lethal mechanisms. For example, defects in homologous recombination (HR) repair arise in cancer cells through inherited or acquired mutations in BRCA1, BRCA2, or other genes in the Fanconi anemia/BRCA pathway, and these tumors have been shown to be particularly sensitive to inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP). Recent work has identified additional genomic and functional assays of DNA repair that provide new predictive and pharmacodynamic biomarkers for these targeted therapies. Here, we examine the development of selective agents targeting DNA repair, including PARP inhibitors; inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM); and inhibitors of classical non-homologous end joining (cNHEJ) and alternative end joining (Alt EJ). We also review the biomarkers that guide the use of these agents and current clinical trials with these therapies.

Profiling Cas9- and Cas12a-induced mutagenesis in Arabidopsis thaliana.

With the advancement of CRISPR technologies, a comprehensive understanding of repair mechanisms following double-strand break (DSB) formation is important for improving the precision and efficiency of genetic modifications. In plant genetics, two Cas nucleases are widely used, i.e. Cas9 and Cas12a, which differ with respect to PAM sequence composition, position of the DSB relative to the PAM, and DSB-end configuration (blunt vs. staggered). The latter difference has led to speculations about different options for repair and recombination. Here, we provide detailed repair profiles for LbCas12a in Arabidopsis thaliana, using identical experimental settings previously reported for Cas9-induced DSBs, thus allowing for a quantitative comparison of both nucleases. For both enzymes, non-homologous end-joining (NHEJ) produces 70% of mutations, whereas polymerase theta-mediated end-joining (TMEJ) generates 30%, indicating that DSB-end configuration does not dictate repair pathway choice. Relevant for genome engineering approaches aimed at integrating exogenous DNA, we found that Cas12a similarly stimulates the integration of T-DNA molecules as does Cas9. Long-read sequencing of both Cas9 and Cas12a repair outcomes further revealed a previously underappreciated degree of DNA loss upon TMEJ. The most notable disparity between Cas9 and Cas12a repair profiles is caused by how NHEJ acts on DSB ends with short overhangs: non-symmetric Cas9 cleavage produce 1 bp insertions, which we here show to depend on polymerase Lambda, whereas staggered Cas12a DSBs are not subjected to fill-in synthesis. We conclude that Cas9 and Cas12a are equally effective for genome engineering purposes, offering flexibility in nuclease choice based on the availability of compatible PAM sequences.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

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.

Spontaneous regression of tumours. Possible cross reactivity of autoantibodies against carbonic anhydrase I.

Spontaneous tumour regression in patients after high dose therapy and autologous stem cell transplantation or patients with standard therapy is accompanied with the presence of high titers autoantibodies against carbonic anhydrase I (CA I). The concomitant presence of aplastic anaemia-like syndrome in these patients points to parallel bone marrow suppression during this period. It seems that CA I, an 'obscure' enzyme, does not have any significant physiological role in humans. One possible explanation points to the fact that autoantibodies against CA I may target another antigen(s) which is(are) important in tumour growth as well as in normal haematopoiesis. One of the candidates for such a target is the DNA polymerase theta.

Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.

Long-read sequence analysis for clustered genomic copy number aberrations revealed architectures of intricately intertwined rearrangements.

Most chromosomal aberrations revealed by chromosomal microarray testing (CMA) are simple; however, very complex chromosomal structural rearrangements can also be found. Although the mechanism of structural rearrangements has been gradually revealed, not all mechanisms have been elucidated. We analyzed the breakpoint-junctions (BJs) of two or more clustered copy number variations (CNVs) in the same chromosome arms to understand their conformation and the mechanism of complex structural rearrangements. Combining CMA with long-read whole-genome sequencing (WGS) analysis, we successfully determined all BJs for the clustered CNVs identified in four patients. Multiple CNVs were intricately intertwined with each other, and clustered CNVs in four patients were involved in global complex chromosomal rearrangements. The BJs of two clustered deletions identified in two patients showed microhomologies, and their characteristics were explained by chromothripsis. In contrast, the BJs in the other two patients, who showed clustered deletions and duplications, consisted of blunt-end and nontemplated insertions. These findings could be explained only by alternative nonhomologous end-joining, a mechanism related to polymerase theta. All the patients had at least one inverted segment. Three patients showed cryptic aberrations involving a disruption and a deletion/duplication, which were not detected by CMA but were first identified by WGS. This result suggested that complex rearrangements should be considered if clustered CNVs are observed in the same chromosome arms. Because CMA has potential limitations in genotype-phenotype correlation analysis, a more detailed analysis by whole genome examination is recommended in cases of suspected complex structural aberrations.

Targeting Polymerase Theta (POLθ) for Cancer Therapy.

Polymerase theta (POLθ) is the critical multi-domain enzyme in microhomology-mediated end-joining DNA double-stranded break repair. POLθ is expressed at low levels in normal tissue but is often overexpressed in cancers, especially in DNA repair deficient cancers, such as homologous-recombination cancers, rendering them exquisitely sensitive to POLθ inhibition secondary to synthetic lethality. Development of POLθ inhibitors is an active area of investigation with inhibitors of the N-terminal helicase domain or the C-terminal polymerase domain currently in clinical trial. Here, we review POLθ-mediated microhomology-mediated end-joining, the development of POLθ inhibitors, and the potential clinical uses of POLθ inhibitors.

Mechanisms of PARP Inhibitor Resistance.

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPi) represent the first medicines based on the targeting of the DNA damage response (DDR). PARPi have become standard of care for first-line maintenance treatment in ovarian cancer and have also been approved in other cancer indications including breast, pancreatic and prostate. Despite their efficacy, resistance to PARPi has been reported clinically and represents a growing patient population with unmet clinical need. Here, we describe the various mechanisms of PARPi resistance that have been identified in pre-clinical models and in the clinic.

Genetic evidence for reconfiguration of DNA polymerase θ active site for error-free translesion synthesis in human cells.

The action mechanisms revealed by the biochemical and structural analyses of replicative and translesion synthesis (TLS) DNA polymerases (Pols) are retained in their cellular roles. In this regard, DNA polymerase θ differs from other Pols in that whereas purified Polθ misincorporates an A opposite 1,N6-ethenodeoxyadenosine (ϵdA) using an abasic-like mode, Polθ performs predominantly error-free TLS in human cells. To test the hypothesis that Polθ adopts a different mechanism for replicating through ϵdA in human cells than in the purified Pol, here we analyze the effects of mutations in the two highly conserved tyrosine residues, Tyr-2387 and Tyr-2391, in the Polθ active site. Our findings that these residues are indispensable for TLS by the purified Pol but are not required in human cells, as well as other findings, provide strong evidence that the Polθ active site is reconfigured in human cells to stabilize ϵdA in the syn conformation for Hoogsteen base pairing with the correct nucleotide. The evidence that a DNA polymerase can configure its active site entirely differently in human cells than in the purified Pol establishes a new paradigm for DNA polymerase function.

Mutational signatures of non-homologous and polymerase theta-mediated end-joining in embryonic stem cells.

Cells employ potentially mutagenic DNA repair mechanisms to avoid the detrimental effects of chromosome breaks on cell survival. While classical non-homologous end-joining (cNHEJ) is largely error-free, alternative end-joining pathways have been described that are intrinsically mutagenic. Which end-joining mechanisms operate in germ and embryonic cells and thus contribute to heritable mutations found in congenital diseases is, however, still largely elusive. Here, we determined the genetic requirements for the repair of CRISPR/Cas9-induced chromosomal breaks of different configurations, and establish the mutational consequences. We find that cNHEJ and polymerase theta-mediated end-joining (TMEJ) act both parallel and redundant in mouse embryonic stem cells and account for virtually all end-joining activity. Surprisingly, mutagenic repair by polymerase theta (Pol θ, encoded by the Polq gene) is most prevalent for blunt double-strand breaks (DSBs), while cNHEJ dictates mutagenic repair of DSBs with protruding ends, in which the cNHEJ polymerases lambda and mu play minor roles. We conclude that cNHEJ-dependent repair of DSBs with protruding ends can explain de novo formation of tandem duplications in mammalian genomes.

Rapid enzymatic synthesis of long RNA polymers: A simple protocol to generate RNA libraries with random sequences.

RNA aptamers have several advantages over DNA aptamers due to their propensity to fold into three-dimensional structures. However, the synthesis of large RNA libraries remains a challenge as it requires more precautions to conserve their functional integrity, especially when such libraries are intended for aptamers or ribozymes selection. Here, we present an enzymatic method that enables the rapid synthesis of RNA polymers thanks to the efficient incorporation of ribonucleotides (NTPs) as well as chemically modified ribonucleotides by human DNA polymerase Theta (θ) mutants. These mutants have the ability to generate long single-stranded RNA polynucleotides of random sequences due to their improved template-free terminal nucleotidyltransferase activity. Here we describe the detailed protocols to produce large and diverse libraries of RNA, to make them ready to use in repeated cycles of Systematic Evolution of Ligands by Exponential enrichment (SELEX) and to synthesize C2'-modified nucleic acids with higher nuclease resistance.

A Distinct Class of Chromoanagenesis Events Characterized by Focal Copy Number Gains.

Chromoanagenesis is the process by which a single catastrophic event creates complex rearrangements confined to a single or a few chromosomes. It is usually characterized by the presence of multiple deletions and/or duplications, as well as by copy neutral rearrangements. In contrast, an array CGH screen of patients with developmental anomalies revealed three patients in which a single chromosome carries from 8 to 11 large copy number gains confined to a single chromosome or chromosomal arm, but the absence of deletions. Subsequent fluorescence in situ hybiridization and massive parallel sequencing revealed the duplicons to be clustered together in distinct locations across the altered chromosomes. Breakpoint junction sequences showed both microhomology and non-templated insertions of up to 40 bp. Hence, these patients each demonstrate a single altered chromosome of clustered insertional duplications, no deletions, and breakpoint junction sequences showing microhomology and/or non-templated insertions. These observations are difficult to reconcile with current mechanistic descriptions of chromothripsis and chromoanasynthesis. Therefore, we hypothesize those rearrangements to be of a mechanistically different origin. In addition, we suggest that large untemplated insertional sequences observed at breakpoints are driven by a non-canonical non-homologous end joining mechanism.

Genetic Control of Replication through N1-methyladenine in Human Cells.

N1-methyl adenine (1-MeA) is formed in DNA by reaction with alkylating agents and naturally occurring methyl halides. The 1-MeA lesion impairs Watson-Crick base pairing and blocks normal DNA replication. Here we identify the translesion synthesis (TLS) DNA polymerases (Pols) required for replicating through 1-MeA in human cells and show that TLS through this lesion is mediated via three different pathways in which Pols ι and θ function in one pathway and Pols η and ζ, respectively, function in the other two pathways. Our biochemical studies indicate that in the Polι/Polθ pathway, Polι would carry out nucleotide insertion opposite 1-MeA from which Polθ would extend synthesis. In the Polη pathway, this Pol alone would function at both the nucleotide insertion and extension steps of TLS, and in the third pathway, Polζ would extend from the nucleotide inserted opposite 1-MeA by an as yet unidentified Pol. Whereas by pushing 1-MeA into the syn conformation and by forming Hoogsteen base pair with the T residue, Polι would carry out TLS opposite 1-MeA, the ability of Polη to replicate through 1-MeA suggests that despite its need for Watson-Crick hydrogen bonding, Polη can stabilize the adduct in its active site. Remarkably, even though Pols η and ι are quite error-prone at inserting nucleotides opposite 1-MeA, TLS opposite this lesion in human cells occurs in a highly error-free fashion. This suggests that the in vivo fidelity of TLS Pols is regulated by factors such as post-translational modifications, protein-protein interactions, and possibly others.

Templated insertions-DNA repair gets acrobatic.

Deletions associated with the repair of DNA double-strand breaks is a source of genetic alternation and a recognized source of disease-causing mutagenesis. Theta-mediated end joining is a DNA repair mechanism, which guarantees deletions by its employment of microhomology (MH) alignment to facilitate end joining. A lesser-characterized templated insertion ability of this pathway, on the other hand, is associated with both deletion and insertion. This mechanism is characterized by at least one round of polymerase θ-mediated synthesis, which does not result in successful repair, followed by a subsequent round of polymerase engagement and synthesis that does lead to repair. Here we focus on the mechanisms by which polymerase θ introduces these insertions-direct, inverse, and a new class which we have termed strand switching. We observe this new class of templated insertions at multiple loci and across multiple species, often at a comparable frequency to those previously characterized. Templated insertion mutations are often enriched in cancer genomes and repeat expansion disorders. This repair mechanism thus contributes to disease-associated mutagenesis, and may plausibly even promote disease. Characterization of the types of polymerase θ-dependent insertions can provide new insight into these diseases and clinical promise for treatment.

Long-lasting, biochemically modified mRNA, and its frameshifted recombinant spike proteins in human tissues and circulation after COVID-19 vaccination.

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.

Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in Arabidopsis thaliana.

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana. We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants.

Somatic mutation patterns at Ig and Non-Ig Loci.

The reverse transcriptase (RT) model of immunoglobulin (Ig) somatic hypermutation (SHM) has received insufficient scientific attention. This is understandable given that DNA deamination mediated by activation-induced deaminase (AID), the initiating step of Ig SHM, has dominated experiments since 2002. We summarise some key history of the RT Ig SHM model dating to 1987. For example, it is now established that DNA polymerase η, the sole DNA repair polymerase involved in post-replication short-patch repair, is an efficient cellular RT. This implies that it is potentially able to initiate target site reverse transcription by RNA-directed DNA repair at AID-induced lesions. Recently, DNA polymerase θ has also been shown to be an efficient cellular RT. Since DNA polymerase θ plays no significant role in Ig SHM, it could serve a similar RNA-dependent DNA polymerase role as DNA polymerase η at non-Ig loci in the putative RNA-templated nucleotide excision repair of bulky adducts and other mutagenic lesions on the transcribed strand. A major yet still poorly recognised consequence of the proposed RT process in Ig SHM is the generation of significant and characteristic strand-biased mutation signatures at both deoxyadenosine/deoxythymidine and deoxyguanosine/deoxycytidine base pairs. In this historical perspective, we highlight how diagnostic strand-biased mutation signatures are detected in vivo during SHM at both Ig loci in germinal centre B lymphocytes and non-Ig loci in cancer genomes. These strand-biased signatures have been significantly obscured by technical issues created by improper use of the polymerase chain reaction technique. A heightened awareness of this fact should contribute to better data interpretation and somatic mutation pattern recognition both at Ig and non-Ig loci.

Transformation and regeneration of DNA polymerase Θ mutant rice plants.

Agrobacterium T-DNA integration into the plant genome is essential for the process of transgenesis and is widely used for genome engineering. The importance of the non-homologous end-joining (NHEJ) protein DNA polymerase Θ, encoded by the PolQ gene, for T-DNA integration is controversial, with some groups claiming it is essential whereas others claim T-DNA integration in Arabidopsis and rice polQ mutant plant tissue. Because of pleiotropic effects of PolQ loss on plant development, scientists have previously had difficulty regenerating transgenic polQ mutant plants. We describe a protocol for regenerating transgenic polQ mutant rice plants using a sequential transformation method. This protocol may be applicable to other plant species.