42 results on '"Pomerantz RT"'
Search Results
2. Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM.
- Author
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Ito F, Li Z, Minakhin L, Chandramouly G, Tyagi M, Betsch R, Krais JJ, Taberi B, Vekariya U, Calbert M, Skorski T, Johnson N, Chen XS, and Pomerantz RT
- Subjects
- Humans, BRCA2 Protein metabolism, BRCA2 Protein genetics, BRCA2 Protein chemistry, BRCA1 Protein metabolism, BRCA1 Protein genetics, BRCA1 Protein chemistry, Piperazines pharmacology, Piperazines chemistry, Cell Line, Tumor, Phthalazines pharmacology, Phthalazines chemistry, Enzyme Inhibitors pharmacology, Enzyme Inhibitors chemistry, Models, Molecular, Adenosine Triphosphatases metabolism, Adenosine Triphosphatases antagonists & inhibitors, Protein Binding, Cryoelectron Microscopy, DNA Helicases metabolism, DNA Helicases chemistry, DNA Helicases genetics, DNA Helicases antagonists & inhibitors, DNA-Directed DNA Polymerase metabolism, DNA-Directed DNA Polymerase chemistry, DNA-Directed DNA Polymerase genetics, DNA Polymerase theta
- Abstract
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 IC
50 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., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
3. PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks.
- Author
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Vekariya U, Minakhin L, Chandramouly G, Tyagi M, Kent T, Sullivan-Reed K, Atkins J, Ralph D, Nieborowska-Skorska M, Kukuyan AM, Tang HY, Pomerantz RT, and Skorski T
- Subjects
- Humans, Poly Adenosine Diphosphate Ribose metabolism, DNA Damage, Animals, DNA-Binding Proteins metabolism, DNA-Binding Proteins genetics, DNA metabolism, DNA genetics, HEK293 Cells, Poly ADP Ribosylation, Poly(ADP-ribose) Polymerases metabolism, Poly(ADP-ribose) Polymerases genetics, Carrier Proteins, Glycoside Hydrolases, Nuclear Proteins, Poly (ADP-Ribose) Polymerase-1 metabolism, Poly (ADP-Ribose) Polymerase-1 genetics, DNA Polymerase theta, DNA End-Joining Repair, DNA-Directed DNA Polymerase metabolism, DNA Breaks, Double-Stranded
- Abstract
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., (© 2024. The Author(s).)
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- 2024
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4. Structural Basis for Polθ-Helicase DNA Binding and Microhomology-Mediated End-Joining.
- Author
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Ito F, Li Z, Minakhin L, Khant HA, Pomerantz RT, and Chen XS
- Abstract
DNA double-strand breaks (DSBs) present a critical threat to genomic integrity, often precipitating genomic instability and oncogenesis. Repair of DSBs predominantly occurs through homologous recombination (HR) and non-homologous end joining (NHEJ). In HR-deficient cells, DNA polymerase theta (Polθ) becomes critical for DSB repair via microhomology-mediated end joining (MMEJ), also termed theta-mediated end joining (TMEJ). Thus, Polθ is synthetically lethal with BRCA1/2 and other HR factors, underscoring its potential as a therapeutic target in HR-deficient cancers. However, the molecular mechanisms governing Polθ-mediated MMEJ remain poorly understood. Here we present a series of cryo-electron microscopy structures of the Polθ helicase domain (Polθ-hel) in complex with DNA containing 3'-overhang. The structures reveal the sequential conformations adopted by Polθ-hel during the critical phases of DNA binding, microhomology searching, and microhomology annealing. The stepwise conformational changes within the Polθ-hel subdomains and its functional dimeric state are pivotal for aligning the 3'-overhangs, facilitating the microhomology search and subsequent annealing necessary for DSB repair via MMEJ. Our findings illustrate the essential molecular switches within Polθ-hel that orchestrate the MMEJ process in DSB repair, laying the groundwork for the development of targeted therapies against the Polθ-hel., Competing Interests: Competing interests X.S.C. is a cofounder of Recombination Therapeutics, LLC. R.T.P. is a cofounder and CSO of Recombination Therapeutics, LLC. The other authors do not declare any competing interests.
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- 2024
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5. 4'-Ethynyl-2'-Deoxycytidine (EdC) Preferentially Targets Lymphoma and Leukemia Subtypes by Inducing Replicative Stress.
- Author
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Calbert ML, Chandramouly G, Adams CM, Saez-Ayala M, Kent T, Tyagi M, Ayyadevara VSSA, Wang Y, Krais JJ, Gordon J, Atkins J, Toma MM, Betzi S, Boghossian AS, Rees MG, Ronan MM, Roth JA, Goldman AR, Gorman N, Mitra R, Childers WE, Graña X, Skorski T, Johnson N, Hurtz C, Morelli X, Eischen CM, and Pomerantz RT
- Subjects
- Humans, Animals, Mice, Cell Line, Tumor, Lymphoma drug therapy, Lymphoma pathology, Lymphoma metabolism, Xenograft Model Antitumor Assays, Leukemia drug therapy, Leukemia pathology, Deoxycytidine Kinase metabolism, Deoxycytidine analogs & derivatives, Deoxycytidine pharmacology
- Abstract
Anticancer nucleosides are effective against solid tumors and hematologic malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induces replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å cocrystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared with FDA-approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a preclinical nucleoside prodrug candidate for DLBCL and ALL., (©2023 American Association for Cancer Research.)
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- 2024
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6. Discovery of a small-molecule inhibitor that traps Polθ on DNA and synergizes with PARP inhibitors.
- Author
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Fried W, Tyagi M, Minakhin L, Chandramouly G, Tredinnick T, Ramanjulu M, Auerbacher W, Calbert M, Rusanov T, Hoang T, Borisonnik N, Betsch R, Krais JJ, Wang Y, Vekariya UM, Gordon J, Morton G, Kent T, Skorski T, Johnson N, Childers W, Chen XS, and Pomerantz RT
- Subjects
- BRCA2 Protein genetics, DNA metabolism, DNA Repair, DNA-Directed DNA Polymerase metabolism, Homologous Recombination, Humans, BRCA1 Protein genetics, Poly(ADP-ribose) Polymerase Inhibitors pharmacology
- Abstract
The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC
50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi., (© 2024. The Author(s).)- Published
- 2024
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7. Genetic separation of Brca1 functions reveal mutation-dependent Polθ vulnerabilities.
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Krais JJ, Glass DJ, Chudoba I, Wang Y, Feng W, Simpson D, Patel P, Liu Z, Neumann-Domer R, Betsch RG, Bernhardy AJ, Bradbury AM, Conger J, Yueh WT, Nacson J, Pomerantz RT, Gupta GP, Testa JR, and Johnson N
- Subjects
- Mice, Animals, Humans, Mutation, Synthetic Lethal Mutations, DNA, BRCA1 Protein genetics, Genes, BRCA2
- Abstract
Homologous recombination (HR)-deficiency induces a dependency on DNA polymerase theta (Polθ/Polq)-mediated end joining, and Polθ inhibitors (Polθi) are in development for cancer therapy. BRCA1 and BRCA2 deficient cells are thought to be synthetic lethal with Polθ, but whether distinct HR gene mutations give rise to equivalent Polθ-dependence, and the events that drive lethality, are unclear. In this study, we utilized mouse models with separate Brca1 functional defects to mechanistically define Brca1-Polθ synthetic lethality. Surprisingly, homozygous Brca1 mutant, Polq
-/- cells were viable, but grew slowly and had chromosomal instability. Brca1 mutant cells proficient in DNA end resection were significantly more dependent on Polθ for viability; here, treatment with Polθi elevated RPA foci, which persisted through mitosis. In an isogenic system, BRCA1 null cells were defective, but PALB2 and BRCA2 mutant cells exhibited active resection, and consequently stronger sensitivity to Polθi. Thus, DNA end resection is a critical determinant of Polθi sensitivity in HR-deficient cells, and should be considered when selecting patients for clinical studies., (© 2023. The Author(s).)- Published
- 2023
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8. Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.
- Author
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Mensah IK, Norvil AB, He M, Lendy E, Hjortland N, Tan H, Pomerantz RT, Mesecar A, and Gowher H
- Subjects
- Drug Development, RNA, RNA-Dependent RNA Polymerase metabolism, Zika Virus enzymology, Gene Expression Profiling, Epigenesis, Genetic, Biotinylation, Protein Structure, Tertiary, Methyltransferases metabolism, Biological Assay methods
- Abstract
RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
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9. Promoter-independent synthesis of chemically modified RNA by human DNA polymerase θ variants.
- Author
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Tredinnick T, Kent T, Minakhin L, Li Z, Madzo J, Chen XS, and Pomerantz RT
- Subjects
- Humans, DNA genetics, Ribonucleotides genetics, Oligonucleotides, DNA Polymerase theta, RNA genetics, DNA-Directed RNA Polymerases genetics
- Abstract
Synthetic RNA oligonucleotides composed of canonical and modified ribonucleotides are highly effective for RNA antisense therapeutics and RNA-based genome engineering applications utilizing CRISPR-Cas9. Yet, synthesis of synthetic RNA using phosphoramidite chemistry is highly inefficient and expensive relative to DNA oligonucleotides, especially for relatively long RNA oligonucleotides. Thus, new biotechnologies are needed to significantly reduce costs, while increasing synthesis rates and yields of synthetic RNA. Here, we engineer human DNA polymerase theta (Polθ) variants and demonstrate their ability to synthesize long (95-200 nt) RNA oligonucleotides with canonical ribonucleotides and ribonucleotide analogs commonly used for stabilizing RNA for therapeutic and genome engineering applications. In contrast to natural promoter-dependent RNA polymerases, Polθ variants synthesize RNA by initiating from DNA or RNA primers, which enables the production of RNA without short abortive byproducts. Remarkably, Polθ variants show the lower capacity to misincorporate ribonucleotides compared to T7 RNA polymerase. Automation of this enzymatic RNA synthesis technology can potentially increase yields while reducing costs of synthetic RNA oligonucleotide production., (© 2023 Tredinnick et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)
- Published
- 2023
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10. Correction: Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.
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Demidova EV, Serebriiskii IG, Vlasenkova R, Kelow S, Andrake MD, Hartman TR, Kent T, Virtucio J, Rosen GL, Pomerantz RT, Dunbrack RL Jr, Golemis EA, Hall MJ, Chen DYT, Daly MB, and Arora S
- Published
- 2023
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11. Author Correction: Polλ promotes microhomology-mediated end-joining.
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Chandramouly G, Jamsen J, Borisonnik N, Tyagi M, Calbert ML, Tredinnick T, Ozdemir AY, Kent T, Demidova EV, Arora S, Wilson SH, and Pomerantz RT
- Published
- 2023
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12. DNA polymerase θ protects leukemia cells from metabolically induced DNA damage.
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Vekariya U, Toma M, Nieborowska-Skorska M, Le BV, Caron MC, Kukuyan AM, Sullivan-Reed K, Podszywalow-Bartnicka P, Chitrala KN, Atkins J, Drzewiecka M, Feng W, Chan J, Chatla S, Golovine K, Jelinek J, Sliwinski T, Ghosh J, Matlawska-Wasowska K, Chandramouly G, Nejati R, Wasik M, Sykes SM, Piwocka K, Hadzijusufovic E, Valent P, Pomerantz RT, Morton G, Childers W, Zhao H, Paietta EM, Levine RL, Tallman MS, Fernandez HF, Litzow MR, Gupta GP, Masson JY, and Skorski T
- Subjects
- Animals, Mice, BRCA2 Protein, DNA metabolism, DNA Polymerase theta, BRCA1 Protein, DNA Damage, Leukemia enzymology, Leukemia genetics
- Abstract
Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect., (© 2023 by The American Society of Hematology.)
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- 2023
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13. Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.
- Author
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Demidova EV, Serebriiskii IG, Vlasenkova R, Kelow S, Andrake MD, Hartman TR, Kent T, Virtucio J, Rosen GL, Pomerantz RT, Dunbrack RL Jr, Golemis EA, Hall MJ, Chen DYT, Daly MB, and Arora S
- Subjects
- Humans, Genetic Predisposition to Disease, DNA Replication, Germ-Line Mutation, Germ Cells, Carcinoma, Renal Cell, Kidney Neoplasms
- Abstract
Background: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined., Methods: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes., Results: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities., Conclusions: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC., (© 2023. The Author(s).)
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- 2023
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14. Polλ promotes microhomology-mediated end-joining.
- Author
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Chandramouly G, Jamsen J, Borisonnik N, Tyagi M, Calbert ML, Tredinnick T, Ozdemir AY, Kent T, Demidova EV, Arora S, Wilson SH, and Pomerantz RT
- Subjects
- Animals, Humans, DNA End-Joining Repair, DNA, Mammals, DNA Breaks, Double-Stranded, DNA Repair
- Abstract
The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2023
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15. DNA Polymerase θ: A Cancer Drug Target with Reverse Transcriptase Activity.
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Chen XS and Pomerantz RT
- Subjects
- Animals, DNA-Directed DNA Polymerase metabolism, Drug Delivery Systems, Humans, DNA Polymerase theta, Antineoplastic Agents pharmacology, DNA-Directed DNA Polymerase drug effects, RNA-Directed DNA Polymerase metabolism
- Abstract
The emergence of precision medicine from the development of Poly (ADP-ribose) polymerase (PARP) inhibitors that preferentially kill cells defective in homologous recombination has sparked wide interest in identifying and characterizing additional DNA repair enzymes that are synthetic lethal with HR factors. DNA polymerase theta (Polθ) is a validated anti-cancer drug target that is synthetic lethal with HR factors and other DNA repair proteins and confers cellular resistance to various genotoxic cancer therapies. Since its initial characterization as a helicase-polymerase fusion protein in 2003, many exciting and unexpected activities of Polθ in microhomology-mediated end-joining (MMEJ) and translesion synthesis (TLS) have been discovered. Here, we provide a short review of Polθ's DNA repair activities and its potential as a drug target and highlight a recent report that reveals Polθ as a naturally occurring reverse transcriptase (RT) in mammalian cells.
- Published
- 2021
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16. Polθ reverse transcribes RNA and promotes RNA-templated DNA repair.
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Chandramouly G, Zhao J, McDevitt S, Rusanov T, Hoang T, Borisonnik N, Treddinick T, Lopezcolorado FW, Kent T, Siddique LA, Mallon J, Huhn J, Shoda Z, Kashkina E, Brambati A, Stark JM, Chen XS, and Pomerantz RT
- Subjects
- Animals, DNA chemistry, DNA Repair, Deoxyribonucleotides, Humans, Mammals genetics, Ribonucleotides, DNA-Directed DNA Polymerase chemistry, RNA
- Abstract
Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Å crystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)
- Published
- 2021
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17. Polθ promotes the repair of 5'-DNA-protein crosslinks by microhomology-mediated end-joining.
- Author
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Chandramouly G, Liao S, Rusanov T, Borisonnik N, Calbert ML, Kent T, Sullivan-Reed K, Vekariya U, Kashkina E, Skorski T, Yan H, and Pomerantz RT
- Subjects
- Animals, Cell Line, DNA chemistry, DNA-Binding Proteins genetics, DNA-Binding Proteins metabolism, DNA-Directed DNA Polymerase deficiency, DNA-Directed DNA Polymerase genetics, Embryonic Stem Cells cytology, Embryonic Stem Cells metabolism, Formaldehyde pharmacology, Humans, Mice, Ovum metabolism, Phosphoric Diester Hydrolases genetics, Phosphoric Diester Hydrolases metabolism, Xenopus growth & development, Xenopus metabolism, DNA Polymerase theta, RNA, Guide, CRISPR-Cas Systems, Cross-Linking Reagents pharmacology, DNA End-Joining Repair drug effects, DNA-Directed DNA Polymerase metabolism
- Abstract
DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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18. Publisher Correction: Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.
- Author
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Black SJ, Ozdemir AY, Kashkina E, Kent T, Rusanov T, Ristic D, Shin Y, Suma A, Hoang T, Chandramouly G, Siddique LA, Borisonnik N, Sullivan-Reed K, Mallon JS, Skorski T, Carnevale V, Murakami KS, Wyman C, and Pomerantz RT
- Abstract
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
- Published
- 2020
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19. Molecular basis of microhomology-mediated end-joining by purified full-length Polθ.
- Author
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Black SJ, Ozdemir AY, Kashkina E, Kent T, Rusanov T, Ristic D, Shin Y, Suma A, Hoang T, Chandramouly G, Siddique LA, Borisonnik N, Sullivan-Reed K, Mallon JS, Skorski T, Carnevale V, Murakami KS, Wyman C, and Pomerantz RT
- Subjects
- Catalytic Domain, DNA Breaks, DNA-Binding Proteins metabolism, DNA-Directed DNA Polymerase genetics, Humans, Models, Molecular, Mutagenesis, Site-Directed, DNA Polymerase theta, DNA Breaks, Double-Stranded, DNA End-Joining Repair physiology, DNA Helicases metabolism, DNA, Single-Stranded metabolism, DNA-Directed DNA Polymerase chemistry, DNA-Directed DNA Polymerase metabolism
- Abstract
DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.
- Published
- 2019
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20. One-step enzymatic modification of RNA 3' termini using polymerase θ.
- Author
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Thomas C, Rusanov T, Hoang T, Augustin T, Kent T, Gaspar I, and Pomerantz RT
- Subjects
- 3' Untranslated Regions genetics, DNA Nucleotidylexotransferase chemistry, DNA Nucleotidylexotransferase metabolism, DNA-Directed DNA Polymerase chemistry, Humans, RNA, Messenger genetics, DNA Polymerase theta, DNA-Directed DNA Polymerase metabolism, RNA, Messenger chemistry, RNA, Messenger metabolism
- Abstract
Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3' terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3' termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30-50 2'-deoxyribonucleotides to the 3' terminus of RNA molecules of various lengths and sequences, and extends RNA 3' termini with an assortment of 2'-deoxy and 2',3'-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3' terminal modification of RNA and potentially enzymatic synthesis of RNA., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2019
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21. Identification of a Small Interface between the Methyltransferase and RNA Polymerase of NS5 that is Essential for Zika Virus Replication.
- Author
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Rusanov T, Kent T, Saeed M, Hoang TM, Thomas C, Rice CM, and Pomerantz RT
- Subjects
- Amino Acid Sequence, Cell Line, Tumor, Humans, Viral Nonstructural Proteins genetics, Zika Virus Infection virology, Methyltransferases genetics, RNA-Dependent RNA Polymerase genetics, Virus Replication genetics, Zika Virus genetics
- Abstract
The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp in vitro. For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.
- Published
- 2018
- Full Text
- View/download PDF
22. Large deletions induced by Cas9 cleavage.
- Author
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Adikusuma F, Piltz S, Corbett MA, Turvey M, McColl SR, Helbig KJ, Beard MR, Hughes J, Pomerantz RT, and Thomas PQ
- Published
- 2018
- Full Text
- View/download PDF
23. Polymerase θ-helicase efficiently unwinds DNA and RNA-DNA hybrids.
- Author
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Ozdemir AY, Rusanov T, Kent T, Siddique LA, and Pomerantz RT
- Subjects
- Amino Acid Sequence, Crystallography, X-Ray, DNA metabolism, DNA Breaks, Double-Stranded, DNA End-Joining Repair physiology, DNA Replication physiology, DNA, Single-Stranded metabolism, DNA-Directed DNA Polymerase physiology, Humans, Nucleic Acid Hybridization, Protein Binding, Recombination, Genetic genetics, DNA Polymerase theta, DNA Helicases metabolism, DNA-Directed DNA Polymerase metabolism
- Abstract
POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1-894) efficiently unwinds DNA with 3'-5' polarity, including DNA with 3' or 5' overhangs, blunt-ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Polθ in replication repair., (© 2018 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2018
- Full Text
- View/download PDF
24. How RNA transcripts coordinate DNA recombination and repair.
- Author
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McDevitt S, Rusanov T, Kent T, Chandramouly G, and Pomerantz RT
- Subjects
- DNA Breaks, Double-Stranded, DNA Repair genetics, DNA Repair physiology, RNA genetics, Rad52 DNA Repair and Recombination Protein genetics, Recombinational DNA Repair genetics, Recombinational DNA Repair physiology, Saccharomyces cerevisiae Proteins genetics, Saccharomyces cerevisiae Proteins metabolism, Rad52 DNA Repair and Recombination Protein metabolism
- Abstract
Genetic studies in yeast indicate that RNA transcripts facilitate homology-directed DNA repair in a manner that is dependent on RAD52. The molecular basis for so-called RNA-DNA repair, however, remains unknown. Using reconstitution assays, we demonstrate that RAD52 directly cooperates with RNA as a sequence-directed ribonucleoprotein complex to promote two related modes of RNA-DNA repair. In a RNA-bridging mechanism, RAD52 assembles recombinant RNA-DNA hybrids that coordinate synapsis and ligation of homologous DNA breaks. In an RNA-templated mechanism, RAD52-mediated RNA-DNA hybrids enable reverse transcription-dependent RNA-to-DNA sequence transfer at DNA breaks that licenses subsequent DNA recombination. Notably, we show that both mechanisms of RNA-DNA repair are promoted by transcription of a homologous DNA template in trans. In summary, these data elucidate how RNA transcripts cooperate with RAD52 to coordinate homology-directed DNA recombination and repair in the absence of a DNA donor, and demonstrate a direct role for transcription in RNA-DNA repair.
- Published
- 2018
- Full Text
- View/download PDF
25. The helicase domain of Polθ counteracts RPA to promote alt-NHEJ.
- Author
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Mateos-Gomez PA, Kent T, Deng SK, McDevitt S, Kashkina E, Hoang TM, Pomerantz RT, and Sfeir A
- Subjects
- Animals, CRISPR-Cas Systems genetics, Cell Line, DNA Breaks, Double-Stranded, Homologous Recombination genetics, Mice, Mice, Inbred C57BL, Mice, Knockout, Replication Protein A genetics, Structure-Activity Relationship, Translocation, Genetic genetics, DNA Polymerase theta, Catalytic Domain genetics, DNA End-Joining Repair genetics, DNA-Directed DNA Polymerase genetics, Embryonic Stem Cells cytology, Replication Protein A antagonists & inhibitors
- Abstract
Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.
- Published
- 2017
- Full Text
- View/download PDF
26. PARP1 restricts Epstein Barr Virus lytic reactivation by binding the BZLF1 promoter.
- Author
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Lupey-Green LN, Moquin SA, Martin KA, McDevitt SM, Hulse M, Caruso LB, Pomerantz RT, Miranda JL, and Tempera I
- Subjects
- Cell Line, Epstein-Barr Virus Infections genetics, Gene Expression Regulation, Viral, Herpesvirus 4, Human genetics, Host-Pathogen Interactions, Humans, Poly (ADP-Ribose) Polymerase-1 genetics, Protein Binding, Trans-Activators metabolism, Virus Latency, Epstein-Barr Virus Infections enzymology, Epstein-Barr Virus Infections virology, Herpesvirus 4, Human physiology, Poly (ADP-Ribose) Polymerase-1 metabolism, Promoter Regions, Genetic, Trans-Activators genetics, Virus Activation
- Abstract
The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
27. Modification of 3' Terminal Ends of DNA and RNA Using DNA Polymerase θ Terminal Transferase Activity.
- Author
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Hoang TM, Kent T, and Pomerantz RT
- Abstract
DNA polymerase θ (Polθ) is a promiscuous enzyme that is essential for the error-prone DNA double-strand break (DSB) repair pathway called alternative end-joining (alt-EJ). During this form of DSB repair, Polθ performs terminal transferase activity at the 3' termini of resected DSBs via templated and non-templated nucleotide addition cycles. Since human Polθ is able to modify the 3' terminal ends of both DNA and RNA with a wide array of large and diverse ribonucleotide and deoxyribonucleotide analogs, its terminal transferase activity is more useful for biotechnology applications than terminal deoxynucleotidyl transferase (TdT). Here, we present in detail simple methods by which purified human Polθ is utilized to modify the 3' terminal ends of RNA and DNA for various applications in biotechnology and biomedical research.
- Published
- 2017
- Full Text
- View/download PDF
28. DNA polymerase θ specializes in incorporating synthetic expanded-size (xDNA) nucleotides.
- Author
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Kent T, Rusanov TD, Hoang TM, Velema WA, Krueger AT, Copeland WC, Kool ET, and Pomerantz RT
- Subjects
- Humans, Nucleotides metabolism, Protein Binding, DNA Polymerase theta, DNA genetics, DNA metabolism, DNA Replication, DNA-Directed DNA Polymerase metabolism
- Abstract
DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)
- Published
- 2016
- Full Text
- View/download PDF
29. DNA Polymerase θ: A Unique Multifunctional End-Joining Machine.
- Author
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Black SJ, Kashkina E, Kent T, and Pomerantz RT
- Abstract
The gene encoding DNA polymerase θ (Polθ) was discovered over ten years ago as having a role in suppressing genome instability in mammalian cells. Studies have now clearly documented an essential function for this unique A-family polymerase in the double-strand break (DSB) repair pathway alternative end-joining (alt-EJ), also known as microhomology-mediated end-joining (MMEJ), in metazoans. Biochemical and cellular studies show that Polθ exhibits a unique ability to perform alt-EJ and during this process the polymerase generates insertion mutations due to its robust terminal transferase activity which involves template-dependent and independent modes of DNA synthesis. Intriguingly, the POLQ gene also encodes for a conserved superfamily 2 Hel308-type ATP-dependent helicase domain which likely assists in alt-EJ and was reported to suppress homologous recombination (HR) via its anti-recombinase activity. Here, we review our current knowledge of Polθ-mediated end-joining, the specific activities of the polymerase and helicase domains, and put into perspective how this multifunctional enzyme promotes alt-EJ repair of DSBs formed during S and G2 cell cycle phases., Competing Interests: R.T.P. has filed provisional patents on the use of Polθ for modifying the 3’ ends of nucleic acids and expanded-size nucleotide analogs as Polθ inhibitors and cancer therapeutics.
- Published
- 2016
- Full Text
- View/download PDF
30. Polymerase θ is a robust terminal transferase that oscillates between three different mechanisms during end-joining.
- Author
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Kent T, Mateos-Gomez PA, Sfeir A, and Pomerantz RT
- Subjects
- Animals, Coenzymes metabolism, Embryonic Stem Cells, Manganese metabolism, Mice, DNA Polymerase theta, DNA metabolism, DNA End-Joining Repair, DNA Nucleotidylexotransferase metabolism, DNA-Directed DNA Polymerase metabolism
- Abstract
DNA polymerase θ (Polθ) promotes insertion mutations during alternative end-joining (alt-EJ) by an unknown mechanism. Here, we discover that mammalian Polθ transfers nucleotides to the 3' terminus of DNA during alt-EJ in vitro and in vivo by oscillating between three different modes of terminal transferase activity: non-templated extension, templated extension in cis, and templated extension in trans. This switching mechanism requires manganese as a co-factor for Polθ template-independent activity and allows for random combinations of templated and non-templated nucleotide insertions. We further find that Polθ terminal transferase activity is most efficient on DNA containing 3' overhangs, is facilitated by an insertion loop and conserved residues that hold the 3' primer terminus, and is surprisingly more proficient than terminal deoxynucleotidyl transferase. In summary, this report identifies an unprecedented switching mechanism used by Polθ to generate genetic diversity during alt-EJ and characterizes Polθ as among the most proficient terminal transferases known.
- Published
- 2016
- Full Text
- View/download PDF
31. Small-Molecule Disruption of RAD52 Rings as a Mechanism for Precision Medicine in BRCA-Deficient Cancers.
- Author
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Chandramouly G, McDevitt S, Sullivan K, Kent T, Luz A, Glickman JF, Andrake M, Skorski T, and Pomerantz RT
- Subjects
- Allosteric Regulation, Apoptosis drug effects, BRCA1 Protein deficiency, BRCA2 Protein deficiency, Cell Line, Cell Proliferation drug effects, DNA Damage drug effects, DNA, Single-Stranded chemistry, DNA, Single-Stranded metabolism, Dihydroxyphenylalanine analogs & derivatives, Dihydroxyphenylalanine chemistry, Dihydroxyphenylalanine metabolism, Dihydroxyphenylalanine toxicity, Electrophoretic Mobility Shift Assay, Humans, Inhibitory Concentration 50, Microscopy, Fluorescence, Neoplasms genetics, Neoplasms metabolism, Neoplasms pathology, Protein Binding, RNA Interference, RNA, Small Interfering metabolism, Rad52 DNA Repair and Recombination Protein genetics, Rad52 DNA Repair and Recombination Protein metabolism, Recombinant Proteins biosynthesis, Recombinant Proteins chemistry, Recombinant Proteins isolation & purification, Small Molecule Libraries metabolism, Small Molecule Libraries toxicity, BRCA1 Protein genetics, BRCA2 Protein genetics, Rad52 DNA Repair and Recombination Protein antagonists & inhibitors, Small Molecule Libraries chemistry
- Abstract
Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
32. Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ.
- Author
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Kent T, Chandramouly G, McDevitt SM, Ozdemir AY, and Pomerantz RT
- Subjects
- Cell Line, Tumor, DNA Breaks, Double-Stranded, DNA-Directed DNA Polymerase chemistry, Humans, Models, Molecular, DNA Polymerase theta, DNA End-Joining Repair physiology, DNA-Directed DNA Polymerase physiology, Models, Genetic
- Abstract
Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break-repair pathway that uses sequence microhomology to recombine broken DNA. Although MMEJ has been implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3' single-strand DNA overhangs with ≥2 bp of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing, then uses the opposing overhang as a template in trans to stabilize the DNA synapse. Polθ exhibits a preference for DNA containing a 5'-terminal phosphate, similarly to polymerases involved in nonhomologous end-joining. Finally, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ that probably promote DNA synapse formation.
- Published
- 2015
- Full Text
- View/download PDF
33. DNA polymerases are error-prone at RecA-mediated recombination intermediates.
- Author
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Pomerantz RT, Goodman MF, and O'Donnell ME
- Subjects
- DNA genetics, DNA Replication physiology, Models, Genetic, Rec A Recombinases genetics, DNA Damage genetics, DNA Polymerase beta metabolism, DNA Replication genetics, Mutagenesis genetics, Rec A Recombinases metabolism, Recombination, Genetic genetics
- Abstract
Genetic studies have suggested that Y-family translesion DNA polymerase IV (DinB) performs error-prone recombination-directed replication (RDR) under conditions of stress due to its ability to promote mutations during double-strand break (DSB) repair in growth-limited E. coli cells. In recent studies we have demonstrated that pol IV is preferentially recruited to D-loop recombination intermediates at stress-induced concentrations and is highly mutagenic during RDR in vitro. These findings verify longstanding genetic data that have implicated pol IV in promoting stress-induced mutagenesis at D-loops. In this Extra View, we demonstrate the surprising finding that A-family pol I, which normally exhibits high-fidelity DNA synthesis, is highly error-prone at D-loops like pol IV. These findings indicate that DNA polymerases are intrinsically error-prone at RecA-mediated D-loops and suggest that auxiliary factors are necessary for suppressing mutations during RDR in non-stressed proliferating cells.
- Published
- 2013
- Full Text
- View/download PDF
34. Preferential D-loop extension by a translesion DNA polymerase underlies error-prone recombination.
- Author
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Pomerantz RT, Kurth I, Goodman MF, and O'Donnell ME
- Subjects
- DNA Polymerase II metabolism, DNA, Bacterial genetics, Escherichia coli metabolism, DNA, Bacterial metabolism, Escherichia coli enzymology, Escherichia coli Proteins metabolism, Recombination, Genetic
- Abstract
Although homologous recombination is considered an accurate form of DNA repair, genetics suggest that the Escherichia coli translesion DNA polymerase IV (Pol IV, also known as DinB) promotes error-prone recombination during stress, which allows cells to overcome adverse conditions. However, how Pol IV functions and is regulated during recombination under stress is unknown. We show that Pol IV is highly proficient in error-prone recombination and is preferentially recruited to displacement loops (D loops) at stress-induced concentrations in vitro. We also found that high-fidelity Pol II switches to exonuclease mode at D loops, which is stimulated by topological stress and reduced deoxyribonucleotide pool concentration during stationary phase. The exonuclease activity of Pol II enables it to compete with Pol IV, which probably suppresses error-prone recombination. These findings indicate that preferential D-loop extension by Pol IV facilitates error-prone recombination and explain how Pol II reduces such errors in vivo.
- Published
- 2013
- Full Text
- View/download PDF
35. Polymerase trafficking: A role for transcription factors in preventing replication fork arrest.
- Author
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Pomerantz RT and O'Donnell M
- Abstract
Replication forks and transcription complexes often collide, which can result in mutagenesis and chromosomal rearrangements. Recent studies of E. coli demonstrate a role for transcription factors in reducing conflicts between replication and transcription. These findings suggest that transcription regulators preserve genome integrity by preventing replication fork arrest.
- Published
- 2010
- Full Text
- View/download PDF
36. What happens when replication and transcription complexes collide?
- Author
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Pomerantz RT and O'Donnell M
- Subjects
- Escherichia coli enzymology, Models, Biological, DNA Replication, DNA-Directed DNA Polymerase metabolism, DNA-Directed RNA Polymerases metabolism, Escherichia coli genetics, Escherichia coli metabolism, Multienzyme Complexes metabolism, Transcription, Genetic
- Abstract
The arrest of replication forks due to collisions with transcription complexes leads to genomic instability and cell death. Mechanisms that promote the progression of replication forks past transcription complexes are therefore essential for propagation and preservation of the genome. Recent studies of E. coli directly investigate the consequences of collisions of the replisome with RNAP polymerase (RNAP) in vitro and provide novel mechanisms by which these encounters may be resolved. Additionally, recent in vivo and in vitro studies support the longstanding hypothesis that auxiliary DNA helicases promote replication through roadblocks such as transcription complexes. Here we review past and recent advances that formulate our current understanding of how the bacterial replisome deals with transcription complexes along the path of chromosome duplication., (© 2010 Landes Bioscience)
- Published
- 2010
- Full Text
- View/download PDF
37. Direct restart of a replication fork stalled by a head-on RNA polymerase.
- Author
-
Pomerantz RT and O'Donnell M
- Subjects
- Bacterial Proteins genetics, Bacterial Proteins metabolism, DNA, Bacterial genetics, Escherichia coli genetics, Escherichia coli Proteins biosynthesis, Escherichia coli Proteins genetics, Transcription Factors genetics, Transcription Factors metabolism, DNA Replication, DNA, Bacterial metabolism, DNA-Directed RNA Polymerases metabolism, Escherichia coli metabolism, Transcription, Genetic
- Abstract
In vivo studies suggest that replication forks are arrested due to encounters with head-on transcription complexes. Yet, the fate of the replisome and RNA polymerase (RNAP) following a head-on collision is unknown. Here, we find that the E. coli replisome stalls upon collision with a head-on transcription complex, but instead of collapsing, the replication fork remains highly stable and eventually resumes elongation after displacing the RNAP from DNA. We also find that the transcription-repair coupling factor, Mfd, promotes direct restart of the fork following the collision by facilitating displacement of the RNAP. These findings demonstrate the intrinsic stability of the replication apparatus and a novel role for the transcription-coupled repair pathway in promoting replication past a RNAP block.
- Published
- 2010
- Full Text
- View/download PDF
38. The replisome uses mRNA as a primer after colliding with RNA polymerase.
- Author
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Pomerantz RT and O'Donnell M
- Subjects
- DNA Replication, DNA, Bacterial metabolism, Escherichia coli genetics, Models, Molecular, DNA Polymerase III metabolism, DNA-Directed RNA Polymerases metabolism, Escherichia coli metabolism, RNA, RNA, Bacterial metabolism, RNA, Messenger metabolism
- Abstract
Replication forks are impeded by DNA damage and protein-nucleic acid complexes such as transcribing RNA polymerase. For example, head-on collision of the replisome with RNA polymerase results in replication fork arrest. However, co-directional collision of the replisome with RNA polymerase has little or no effect on fork progression. Here we examine co-directional collisions between a replisome and RNA polymerase in vitro. We show that the Escherichia coli replisome uses the RNA transcript as a primer to continue leading-strand synthesis after the collision with RNA polymerase that is displaced from the DNA. This action results in a discontinuity in the leading strand, yet the replisome remains intact and bound to DNA during the entire process. These findings underscore the notable plasticity by which the replisome operates to circumvent obstacles in its path and may explain why the leading strand is synthesized discontinuously in vivo.
- Published
- 2008
- Full Text
- View/download PDF
39. Replisome mechanics: insights into a twin DNA polymerase machine.
- Author
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Pomerantz RT and O'Donnell M
- Subjects
- DNA-Directed DNA Polymerase chemistry, Escherichia coli genetics, Escherichia coli metabolism, Models, Biological, Models, Molecular, DNA Replication genetics, DNA-Directed DNA Polymerase metabolism
- Abstract
Chromosomal replicases are multicomponent machines that copy DNA with remarkable speed and processivity. The organization of the replisome reveals a twin DNA polymerase design ideally suited for concurrent synthesis of leading and lagging strands. Recent structural and biochemical studies of Escherichia coli and eukaryotic replication components provide intricate details of the organization and inner workings of cellular replicases. In particular, studies of sliding clamps and clamp-loader subunits elucidate the mechanisms of replisome processivity and lagging strand synthesis. These studies demonstrate close similarities between the bacterial and eukaryotic replication machineries.
- Published
- 2007
- Full Text
- View/download PDF
40. A mechanism of nucleotide misincorporation during transcription due to template-strand misalignment.
- Author
-
Pomerantz RT, Temiakov D, Anikin M, Vassylyev DG, and McAllister WT
- Subjects
- Base Pair Mismatch, Base Sequence, Binding Sites, DNA Repair, DNA-Directed DNA Polymerase metabolism, DNA-Directed RNA Polymerases chemistry, Escherichia coli genetics, Frameshift Mutation, Gene Deletion, Models, Genetic, Molecular Sequence Data, Protein Conformation, Time Factors, Viral Proteins chemistry, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism, Transcription, Genetic, Viral Proteins genetics, Viral Proteins metabolism
- Abstract
Transcription errors by T7 RNA polymerase (RNAP) may occur as the result of a mechanism in which the template base two positions downstream of the 3' end of the RNA (the TSn+1 base) is utilized during two consecutive nucleotide-addition cycles. In the first cycle, misalignment of the template strand leads to incorporation of a nucleotide that is complementary to the TSn+1 base. In the second cycle, the template is realigned and the mismatched primer is efficiently extended, resulting in a substitution error. Proper organization of the transcription bubble is required for maintaining the correct register of the DNA template, as the presence of a complementary nontemplate strand opposite the TSn+1 base suppresses template misalignment. Our findings for T7 RNAP are in contrast to related DNA polymerases of the Pol I type, which fail to extend mismatches efficiently and generate predominantly deletion errors as a result of template-strand misalignment.
- Published
- 2006
- Full Text
- View/download PDF
41. Template misalignment in multisubunit RNA polymerases and transcription fidelity.
- Author
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Kashkina E, Anikin M, Brueckner F, Pomerantz RT, McAllister WT, Cramer P, and Temiakov D
- Subjects
- Base Sequence, Binding Sites, Binding, Competitive, DNA chemistry, Escherichia coli enzymology, Models, Genetic, Molecular Sequence Data, Saccharomyces cerevisiae enzymology, Spectrometry, Fluorescence, Thermus enzymology, Time Factors, DNA-Directed RNA Polymerases chemistry, DNA-Directed RNA Polymerases genetics, Transcription, Genetic, Viral Proteins chemistry
- Abstract
Recent work showed that the single-subunit T7 RNA polymerase (RNAP) can generate misincorporation errors by a mechanism that involves misalignment of the DNA template strand. Here, we show that the same mechanism can produce errors during transcription by the multisubunit yeast RNAP II and bacterial RNAPs. Fluorescence spectroscopy reveals a reorganization of the template strand during this process, and molecular modeling suggests an open space above the polymerase active site that could accommodate a misaligned base. Substrate competition assays indicate that template misalignment, not misincorporation, is the preferred mechanism for substitution errors by cellular RNAPs. Misalignment could account for data previously taken as evidence for additional NTP binding sites downstream of the active site. Analysis of the effects of different template topologies on misincorporation indicates that the duplex DNA immediately downstream of the active site plays an important role in transcription fidelity.
- Published
- 2006
- Full Text
- View/download PDF
42. A tightly regulated molecular motor based upon T7 RNA polymerase.
- Author
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Pomerantz RT, Ramjit R, Gueroui Z, Place C, Anikin M, Leuba S, Zlatanova J, and McAllister WT
- Subjects
- Base Sequence, Enzymes, Immobilized, Models, Molecular, RNA genetics, RNA metabolism, DNA-Directed RNA Polymerases chemistry, DNA-Directed RNA Polymerases metabolism, Molecular Motor Proteins chemistry, Molecular Motor Proteins metabolism, Nanotechnology, Viral Proteins chemistry, Viral Proteins metabolism
- Abstract
Controlled movement of materials or molecules within the nanometer range is essential in many applications of nanotechnology. Here we report the capture, movement, and release of cargo molecules along DNA by a modified form of T7 RNA polymerase (RNAP) in a manner that is controlled by the sequence of the DNA. Using single-molecule methods, we visualize the assembly and manipulation of nanodevices and the ability to harness rotary and linear forces of the RNAP motor.
- Published
- 2005
- Full Text
- View/download PDF
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