{"id":{"repo_id":"milano","oai_identifier":"oai:air.unimi.it:2434/1016672"},"canonical_url":"https://search.dev.ndltd.org/etd/milano/oai:air.unimi.it:2434/1016672","repository":{"repo_id":"milano","name":"Università degli Studi di Milano","base_url":"https://air.unimi.it/oai/request"},"display":{"title":"UNRAVELING THE MOLECULAR MECHANISM OF BRCA2-POLTETA INTERPLAY IN PREVENTING SSDNA GAP ACCUMULATION DURING DNA REPLICATION","abstract":"DNA Polymerase theta (POLθ), encoded by POLQ, plays a critical role in alternative non-homologous end joining (alt-NHEJ), a backup pathway for repairing DNA double-strand breaks in the absence of homologous recombination. The synthetic lethal relationship between POLQ and BRCA1/2 has been well-established, but the underlying molecular mechanisms remain poorly understood. Here, we investigate the role of POLθ beyond alt-NHEJ and its interplay with BRCA2 during DNA replication. We find that BRCA2-deficient cells accumulate replication-associated single-stranded DNA (ssDNA) gaps, which lead us to explore the role of POLθ in repairing these gaps. By employing a novel POLθ polymerase inhibitor (POLθi), we reveal the crucial function of POLθ in filling ssDNA gaps. Treatment of BRCA1/2-deficient cells with POLθi induces DNA damage, as indicated by the accumulation of γH2AX. Through electron microscopy analysis of replication intermediates, we identify fork breakage as the primary source of DNA damage and we find that MRE11-NBS1-CtIP nuclease is responsible for this process. We further investigate the origin of ssDNA gaps and find that they mainly arise from SMUG1 processing of demethylation intermediates, such as 5hmC, leading to the generation of abasic (AP) sites. Additionally, we demonstrate that the accumulation of AP sites impedes fork progression, resulting in ssDNA formation through repriming ahead of the lesion by PRIMPOL. Notably, we found that POLθ fills these ssDNA gaps, and its absence, along with BRCA2, results in massive DNA damage and cell death. Moreover, APEX1-deficient cells, exhibiting increased endogenous AP sites, display ssDNA gaps and sensitivity to the loss of BRCA2 and POLθ. Our findings unravel the molecular mechanisms underlying the synthetic lethal interaction between BRCA2 and POLQ during DNA replication. The heightened vulnerability of BRCA2-deficient or mutated tumors to the accumulation of ssDNA gaps highlights the potential of targeting these gaps as therapeutic strategies, offering promising avenues for precision cancer treatments.","abstract_html":"DNA Polymerase theta (POLθ), encoded by POLQ, plays a critical role in alternative non-homologous end joining (alt-NHEJ), a backup pathway for repairing DNA double-strand breaks in the absence of homologous recombination. The synthetic lethal relationship between POLQ and BRCA1/2 has been well-established, but the underlying molecular mechanisms remain poorly understood. Here, we investigate the role of POLθ beyond alt-NHEJ and its interplay with BRCA2 during DNA replication. We find that BRCA2-deficient cells accumulate replication-associated single-stranded DNA (ssDNA) gaps, which lead us to explore the role of POLθ in repairing these gaps. By employing a novel POLθ polymerase inhibitor (POLθi), we reveal the crucial function of POLθ in filling ssDNA gaps. Treatment of BRCA1/2-deficient cells with POLθi induces DNA damage, as indicated by the accumulation of γH2AX. Through electron microscopy analysis of replication intermediates, we identify fork breakage as the primary source of DNA damage and we find that MRE11-NBS1-CtIP nuclease is responsible for this process. We further investigate the origin of ssDNA gaps and find that they mainly arise from SMUG1 processing of demethylation intermediates, such as 5hmC, leading to the generation of abasic (AP) sites. Additionally, we demonstrate that the accumulation of AP sites impedes fork progression, resulting in ssDNA formation through repriming ahead of the lesion by PRIMPOL. Notably, we found that POLθ fills these ssDNA gaps, and its absence, along with BRCA2, results in massive DNA damage and cell death. Moreover, APEX1-deficient cells, exhibiting increased endogenous AP sites, display ssDNA gaps and sensitivity to the loss of BRCA2 and POLθ. Our findings unravel the molecular mechanisms underlying the synthetic lethal interaction between BRCA2 and POLQ during DNA replication. The heightened vulnerability of BRCA2-deficient or mutated tumors to the accumulation of ssDNA gaps highlights the potential of targeting these gaps as therapeutic strategies, offering promising avenues for precision cancer treatments.","abstract_has_math":false,"creators":["RAMIREZ OTERO, MIGUEL ANGEL"],"institution":"Università degli Studi di Milano","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":null,"school":null,"contributors":["internal advisor: F.d'Adda di Fagagna ; tutor: V. Costanzo ; phd coordinator: S. Minucci","M.A. RAMIREZ OTERO","COSTANZO, VINCENZO","MINUCCI, SAVERIO"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2023,"date_issued":"2023-12-12","date_published":"2023-12-12","updated_at":"2026-07-27T20:19:05Z","subjects":["DNA replication","POL theta","BRCA2","DNA replication stress","Settore BIO/10 - Biochimica"],"languages":["eng"],"rights":["info:eu-repo/semantics/openAccess"],"rights_urls":[],"identifier_entries":[{"key":"dc:identifier","label":"Identifier","values":["http://dx.doi.org/10.13130/ramirez-otero-miguel-angel_phd2023-12-12","10.13130/ramirez-otero-miguel-angel_phd2023-12-12"],"render_values":[{"text":"http://dx.doi.org/10.13130/ramirez-otero-miguel-angel_phd2023-12-12","href":"http://dx.doi.org/10.13130/ramirez-otero-miguel-angel_phd2023-12-12","code":true},{"text":"10.13130/ramirez-otero-miguel-angel_phd2023-12-12","href":"https://doi.org/10.13130/ramirez-otero-miguel-angel_phd2023-12-12","code":true}]}]},"links":{"outbound_url":"https://hdl.handle.net/2434/1016672","outbound_label":"Handle","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["internal advisor: F.d'Adda di Fagagna ; tutor: V. 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The synthetic lethal relationship between POLQ and BRCA1/2 has been well-established, but the underlying molecular mechanisms remain poorly understood. Here, we investigate the role of POLθ beyond alt-NHEJ and its interplay with BRCA2 during DNA replication. We find that BRCA2-deficient cells accumulate replication-associated single-stranded DNA (ssDNA) gaps, which lead us to explore the role of POLθ in repairing these gaps. By employing a novel POLθ polymerase inhibitor (POLθi), we reveal the crucial function of POLθ in filling ssDNA gaps. Treatment of BRCA1/2-deficient cells with POLθi induces DNA damage, as indicated by the accumulation of γH2AX. Through electron microscopy analysis of replication intermediates, we identify fork breakage as the primary source of DNA damage and we find that MRE11-NBS1-CtIP nuclease is responsible for this process. We further investigate the origin of ssDNA gaps and find that they mainly arise from SMUG1 processing of demethylation intermediates, such as 5hmC, leading to the generation of abasic (AP) sites. Additionally, we demonstrate that the accumulation of AP sites impedes fork progression, resulting in ssDNA formation through repriming ahead of the lesion by PRIMPOL. Notably, we found that POLθ fills these ssDNA gaps, and its absence, along with BRCA2, results in massive DNA damage and cell death. Moreover, APEX1-deficient cells, exhibiting increased endogenous AP sites, display ssDNA gaps and sensitivity to the loss of BRCA2 and POLθ. Our findings unravel the molecular mechanisms underlying the synthetic lethal interaction between BRCA2 and POLQ during DNA replication. The heightened vulnerability of BRCA2-deficient or mutated tumors to the accumulation of ssDNA gaps highlights the potential of targeting these gaps as therapeutic strategies, offering promising avenues for precision cancer treatments."]},{"key":"dc:title","label":"Title","values":["UNRAVELING THE MOLECULAR MECHANISM OF BRCA2-POLTETA INTERPLAY IN PREVENTING SSDNA GAP ACCUMULATION DURING DNA REPLICATION"]}]}],"canonical_facts":{"dc:contributor":["internal advisor: F.d'Adda di Fagagna ; tutor: V. Costanzo ; phd coordinator: S. Minucci","M.A. RAMIREZ OTERO","COSTANZO, VINCENZO","MINUCCI, SAVERIO"],"dc:creator":["RAMIREZ OTERO, MIGUEL ANGEL"],"dc:date":["2023-12-12"],"dc:description":["DNA Polymerase theta (POLθ), encoded by POLQ, plays a critical role in alternative non-homologous end joining (alt-NHEJ), a backup pathway for repairing DNA double-strand breaks in the absence of homologous recombination. The synthetic lethal relationship between POLQ and BRCA1/2 has been well-established, but the underlying molecular mechanisms remain poorly understood. Here, we investigate the role of POLθ beyond alt-NHEJ and its interplay with BRCA2 during DNA replication. We find that BRCA2-deficient cells accumulate replication-associated single-stranded DNA (ssDNA) gaps, which lead us to explore the role of POLθ in repairing these gaps. By employing a novel POLθ polymerase inhibitor (POLθi), we reveal the crucial function of POLθ in filling ssDNA gaps. Treatment of BRCA1/2-deficient cells with POLθi induces DNA damage, as indicated by the accumulation of γH2AX. Through electron microscopy analysis of replication intermediates, we identify fork breakage as the primary source of DNA damage and we find that MRE11-NBS1-CtIP nuclease is responsible for this process. We further investigate the origin of ssDNA gaps and find that they mainly arise from SMUG1 processing of demethylation intermediates, such as 5hmC, leading to the generation of abasic (AP) sites. Additionally, we demonstrate that the accumulation of AP sites impedes fork progression, resulting in ssDNA formation through repriming ahead of the lesion by PRIMPOL. Notably, we found that POLθ fills these ssDNA gaps, and its absence, along with BRCA2, results in massive DNA damage and cell death. Moreover, APEX1-deficient cells, exhibiting increased endogenous AP sites, display ssDNA gaps and sensitivity to the loss of BRCA2 and POLθ. Our findings unravel the molecular mechanisms underlying the synthetic lethal interaction between BRCA2 and POLQ during DNA replication. The heightened vulnerability of BRCA2-deficient or mutated tumors to the accumulation of ssDNA gaps highlights the potential of targeting these gaps as therapeutic strategies, offering promising avenues for precision cancer treatments."],"dc:identifier":["https://hdl.handle.net/2434/1016672","http://dx.doi.org/10.13130/ramirez-otero-miguel-angel_phd2023-12-12","10.13130/ramirez-otero-miguel-angel_phd2023-12-12"],"dc:language":["eng"],"dc:publisher":["Università degli Studi di Milano"],"dc:rights":["info:eu-repo/semantics/openAccess"],"dc:subject":["DNA replication","POL theta","BRCA2","DNA replication stress","Settore BIO/10 - Biochimica"],"dc:title":["UNRAVELING THE MOLECULAR MECHANISM OF BRCA2-POLTETA INTERPLAY IN PREVENTING SSDNA GAP ACCUMULATION DURING DNA REPLICATION"],"dc:type":["info:eu-repo/semantics/doctoralThesis"]},"updated_at":"2026-07-27T20:19:05Z"}