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postgraduate thesis: Mechanisms of ultrafine anaphase bridge (UFB) binding proteins, RIF1 and PP1, in regulating UFB resolution
Title | Mechanisms of ultrafine anaphase bridge (UFB) binding proteins, RIF1 and PP1, in regulating UFB resolution |
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Authors | |
Issue Date | 2023 |
Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
Citation | Kong, N. [孔楠楠]. (2023). Mechanisms of ultrafine anaphase bridge (UFB) binding proteins, RIF1 and PP1, in regulating UFB resolution. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. |
Abstract | Chromosome segregation is a crucial step in producing identical daughter cells during cell division, whose defects can induce genome instability or chromosomal aberrations. Ultrafine anaphase bridges (UFBs) are intertwined DNA structures between disjoining sister chromatids persistent in anaphase that can pose threats to faithful chromosome segregation. Elevated frequency of UFBs is observed in many cancer cells and impaired UFB resolution in mitosis can induce DNA damage and aneuploidy. Thus, we aim to investigate the detailed mechanisms of how UFBs are processed in mitosis.
Until now, multiple UFB-associated factors have been identified to be involved in the resolution of UFBs. PLK1-interacting checkpoint helicase (PICH) acts as the main recruiting factor to recruit other factors, including Bloom’s syndrome helicase (BLM) and Rap1 interacting factor 1 (RIF1), onto UFBs. RIF1 was reported to localize on UFBs dependent on PICH but independent of BLM. RIF1 ensures timely resolution of UFBs and sister chromatid disjunction. Therefore, we investigate the detailed mechanism of how RIF1 regulates UFB resolution. We first revealed that RIF1 safeguards faithful chromosome segregation by inhibiting DNA breakage. 53BP1 nuclear bodies and micronuclei can be induced when RIF1 is depleted. Interestingly, RIF1 possibly works in distinct pathways with BLM on UFB resolution to some extent due to the additive effect observed upon their co-depletion.
To study the specific function of RIF1 involved in mitosis, the auxin-inducible degron system was employed to assess the immediate consequences after RIF1 rapid depletion. Together with the synchronization methods, we specifically depleted RIF1 in the G2/M phase and successfully attributed that RIF1 indeed functions in the G2/M phase to prevent chromosomal instability. We also showed that RIF1 localizes on PICH-positive double-stranded UFBs but is excluded from RPA-positive single-stranded UFBs. More importantly, RIF1 prevents the formation of single-stranded RPA-coated UFBs derived from BLM helicase. Thus, we proposed that RIF1 antagonizes either BLM activity or its localization on UFBs.
With the study of RIF1 truncations or mutants, we revealed that PP1 interaction and UFB localization are both mediated by the CI domain of RIF1. Depletion of PP1 phenocopies RIF1 depletion in promoting the formation of BLM- or RPA-positive UFBs. Importantly, we showed that RIF1-PP1 inhibits the interaction between PICH and BLM on UFBs by mediating their dephosphorylation, which caused the exclusion of BLM from UFBs and reduced formation of single-stranded UFBs. This is also validated by the phosphorylation-resistant mutant of PICH, of which its expression resulted in reduced PICH-BLM interaction and decreased formation of single-stranded UFBs. Therefore, we clarified PP1 as a novel UFB-associated factor recruited by RIF1 to play a role in UFB resolution. On the other hand, RIF1-PP1 interaction is inhibited by CDK1-mediated phosphorylation at the CIII domain of RIF1 in prometaphase.
Overall, our study reveals a detailed mechanism of how RIF1 facilitates the resolution of the persistent UFBs to maintain genome stability. We uncover its regulating roles in UFB resolution via its downstream effector PP1. |
Degree | Doctor of Philosophy |
Subject | DNA-binding proteins Phosphoprotein phosphatases Cell division |
Dept/Program | Biological Sciences |
Persistent Identifier | http://hdl.handle.net/10722/336602 |
DC Field | Value | Language |
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dc.contributor.author | Kong, Nannan | - |
dc.contributor.author | 孔楠楠 | - |
dc.date.accessioned | 2024-02-26T08:30:35Z | - |
dc.date.available | 2024-02-26T08:30:35Z | - |
dc.date.issued | 2023 | - |
dc.identifier.citation | Kong, N. [孔楠楠]. (2023). Mechanisms of ultrafine anaphase bridge (UFB) binding proteins, RIF1 and PP1, in regulating UFB resolution. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. | - |
dc.identifier.uri | http://hdl.handle.net/10722/336602 | - |
dc.description.abstract | Chromosome segregation is a crucial step in producing identical daughter cells during cell division, whose defects can induce genome instability or chromosomal aberrations. Ultrafine anaphase bridges (UFBs) are intertwined DNA structures between disjoining sister chromatids persistent in anaphase that can pose threats to faithful chromosome segregation. Elevated frequency of UFBs is observed in many cancer cells and impaired UFB resolution in mitosis can induce DNA damage and aneuploidy. Thus, we aim to investigate the detailed mechanisms of how UFBs are processed in mitosis. Until now, multiple UFB-associated factors have been identified to be involved in the resolution of UFBs. PLK1-interacting checkpoint helicase (PICH) acts as the main recruiting factor to recruit other factors, including Bloom’s syndrome helicase (BLM) and Rap1 interacting factor 1 (RIF1), onto UFBs. RIF1 was reported to localize on UFBs dependent on PICH but independent of BLM. RIF1 ensures timely resolution of UFBs and sister chromatid disjunction. Therefore, we investigate the detailed mechanism of how RIF1 regulates UFB resolution. We first revealed that RIF1 safeguards faithful chromosome segregation by inhibiting DNA breakage. 53BP1 nuclear bodies and micronuclei can be induced when RIF1 is depleted. Interestingly, RIF1 possibly works in distinct pathways with BLM on UFB resolution to some extent due to the additive effect observed upon their co-depletion. To study the specific function of RIF1 involved in mitosis, the auxin-inducible degron system was employed to assess the immediate consequences after RIF1 rapid depletion. Together with the synchronization methods, we specifically depleted RIF1 in the G2/M phase and successfully attributed that RIF1 indeed functions in the G2/M phase to prevent chromosomal instability. We also showed that RIF1 localizes on PICH-positive double-stranded UFBs but is excluded from RPA-positive single-stranded UFBs. More importantly, RIF1 prevents the formation of single-stranded RPA-coated UFBs derived from BLM helicase. Thus, we proposed that RIF1 antagonizes either BLM activity or its localization on UFBs. With the study of RIF1 truncations or mutants, we revealed that PP1 interaction and UFB localization are both mediated by the CI domain of RIF1. Depletion of PP1 phenocopies RIF1 depletion in promoting the formation of BLM- or RPA-positive UFBs. Importantly, we showed that RIF1-PP1 inhibits the interaction between PICH and BLM on UFBs by mediating their dephosphorylation, which caused the exclusion of BLM from UFBs and reduced formation of single-stranded UFBs. This is also validated by the phosphorylation-resistant mutant of PICH, of which its expression resulted in reduced PICH-BLM interaction and decreased formation of single-stranded UFBs. Therefore, we clarified PP1 as a novel UFB-associated factor recruited by RIF1 to play a role in UFB resolution. On the other hand, RIF1-PP1 interaction is inhibited by CDK1-mediated phosphorylation at the CIII domain of RIF1 in prometaphase. Overall, our study reveals a detailed mechanism of how RIF1 facilitates the resolution of the persistent UFBs to maintain genome stability. We uncover its regulating roles in UFB resolution via its downstream effector PP1. | - |
dc.language | eng | - |
dc.publisher | The University of Hong Kong (Pokfulam, Hong Kong) | - |
dc.relation.ispartof | HKU Theses Online (HKUTO) | - |
dc.rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works. | - |
dc.rights | This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. | - |
dc.subject.lcsh | DNA-binding proteins | - |
dc.subject.lcsh | Phosphoprotein phosphatases | - |
dc.subject.lcsh | Cell division | - |
dc.title | Mechanisms of ultrafine anaphase bridge (UFB) binding proteins, RIF1 and PP1, in regulating UFB resolution | - |
dc.type | PG_Thesis | - |
dc.description.thesisname | Doctor of Philosophy | - |
dc.description.thesislevel | Doctoral | - |
dc.description.thesisdiscipline | Biological Sciences | - |
dc.description.nature | published_or_final_version | - |
dc.date.hkucongregation | 2024 | - |
dc.identifier.mmsid | 991044770600003414 | - |