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Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
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coupling of human DNA excision repair and the DNA Damage Checkpoint in a defined in vitro system
Journal of Biological Chemistry, 2014Co-Authors: Laura A Lindseyboltz, Michael G Kemp, Joyce T Reardon, Vanessa Derocco, Ravi R Iyer, Paul Modrich, Aziz SancarAbstract:DNA repair and DNA Damage Checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA Damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA Damage Checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA Damage Checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA Damaged by a UV-mimetic agent to analyze the basic steps of DNA Damage Checkpoint response in a biochemically defined system. We find that Checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to Damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA Damage Checkpoint response.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Rui Ye, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Abstract Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage. Background: AtCRY2 forms photobodies in plants. Results: AtCRY2 forms photobodies in human cells. By fusing AtCRY2 to a human Checkpoint protein, the DNA Damage response can be activated by light. Conclusion: AtCRY2 and its fusion proteins can achieve light-induced protein multimerization independent of other plant proteins. Significance: AtCRY2 can function as an optogenetic tool to modulate signaling pathways.
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purification and characterization of human DNA Damage Checkpoint rad complexes
Proceedings of the National Academy of Sciences of the United States of America, 2001Co-Authors: Laura A Lindseyboltz, Vladimir P Bermudez, Jerard Hurwitz, Aziz SancarAbstract:Checkpoint Rad proteins function early in the DNA Damage Checkpoint signaling cascade to arrest cell cycle progression in response to DNA Damage. This Checkpoint ensures the transmission of an intact genetic complement to daughter cells. To learn about the Damage sensor function of the human Checkpoint Rad proteins, we purified a heteropentameric complex composed of hRad17-RFCp36-RFCp37-RFCp38-RFCp40 (hRad17-RFC) and a heterotrimeric complex composed of hRad9-hHus1-hRad1 (Checkpoint 9-1-1 complex). hRad17-RFC binds to DNA, with a preference for primed DNA and possesses weak ATPase activity that is stimulated by primed DNA and single-stranded DNA. hRad17-RFC forms a complex with the 9-1-1 heterotrimer reminiscent of the replication factor C/proliferating cell nuclear antigen clamp loader/sliding clamp complex of the replication machinery. These findings constitute biochemical support for models regarding the roles of Checkpoint Rads as Damage sensors in the DNA Damage Checkpoint response of human cells.
Irem Ozkandagliyan - One of the best experts on this subject based on the ideXlab platform.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Rui Ye, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Abstract Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage. Background: AtCRY2 forms photobodies in plants. Results: AtCRY2 forms photobodies in human cells. By fusing AtCRY2 to a human Checkpoint protein, the DNA Damage response can be activated by light. Conclusion: AtCRY2 and its fusion proteins can achieve light-induced protein multimerization independent of other plant proteins. Significance: AtCRY2 can function as an optogenetic tool to modulate signaling pathways.
Lyle A. Simmons - One of the best experts on this subject based on the ideXlab platform.
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ddca antagonizes a bacterial DNA Damage Checkpoint
Molecular Microbiology, 2019Co-Authors: Peter E. Burby, Zackary W. Simmons, Lyle A. SimmonsAbstract:Bacteria coordinate DNA replication and cell division, ensuring a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA Damage, a cell cycle Checkpoint is activated by expressing a cell division inhibitor. The prevailing model is that activation of the DNA Damage response and protease-mediated degradation of the inhibitor is sufficient to regulate the Checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to DNA Damage. Similar to the Checkpoint recovery proteases, the DNA Damage sensitivity resulting from ddcA deletion depends on the Checkpoint enforcement protein YneA. Using several genetic approaches, we show that DdcA function is distinct from the Checkpoint recovery process. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels and stability, further supporting the conclusion that DdcA regulates YneA independent of proteolysis. Using a functional GFP-YneA fusion we found that DdcA prevents YneA-dependent cell elongation independent of YneA localization. Together, our results suggest that DdcA acts by helping to set a threshold of YneA required to establish the cell cycle Checkpoint, uncovering a new regulatory step controlling activation of the DNA Damage Checkpoint in Bacillus subtilis.
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ddca antagonizes a bacterial DNA Damage Checkpoint
bioRxiv, 2018Co-Authors: Peter E. Burby, Zackary W. Simmons, Lyle A. SimmonsAbstract:Bacteria coordinate DNA replication and cell division, ensuring that a complete set of genetic material is passed onto the next generation. When bacteria encounter DNA Damage or impediments to DNA replication, a cell cycle Checkpoint is activated to delay cell division by expressing a cell division inhibitor. The prevailing model for bacterial DNA Damage Checkpoints is that activation of the DNA Damage response and protease mediated degradation of the cell division inhibitor is sufficient to regulate the Checkpoint process. Our recent genome-wide screens identified the gene ddcA as critical for surviving exposure to a broad spectrum of DNA Damage. The ddcA deletion phenotypes are dependent on the Checkpoint enforcement protein YneA. We found that expression of the Checkpoint recovery proteases could not compensate for ddcA deletion. Similarly, expression if ddcA could not compensate for the absence of the Checkpoint recovery proteases, indicating that DdcA function is distinct from the Checkpoint recovery step. Deletion of ddcA resulted in sensitivity to yneA overexpression independent of YneA protein levels or stability, further supporting the conclusion that DdcA regulates YneA through a proteolysis independent mechanism. Using a functional GFP-YneA we found that DdcA inhibits YneA activity independent of YneA localization, suggesting that DdcA may regulate YneA access to its target. These results uncover a regulatory step that is important for controlling the DNA Damage Checkpoint in bacteria, and suggests that the typical mechanism of degrading the Checkpoint enforcement protein is insufficient to control the rate of cell division in response to DNA Damage.
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discovery of a dual protease mechanism that promotes DNA Damage Checkpoint recovery
PLOS Genetics, 2018Co-Authors: Peter E. Burby, Zackary W. Simmons, Jeremy W Schroeder, Lyle A. SimmonsAbstract:The DNA Damage response is a signaling pathway found throughout biology. In many bacteria the DNA Damage Checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair Damaged chromosomes. How cells promote Checkpoint recovery after sensing successful repair is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA Damage Checkpoint recovery in Bacillus subtilis. Deletion of both proteases leads to accumulation of the Checkpoint protein YneA. We show that DNA Damage sensitivity and increased cell elongation in protease mutants depends on yneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that both proteases interact with YneA and that one of the two proteases, CtpA, directly cleaves YneA in vitro. With these results, we report the mechanism for DNA Damage Checkpoint recovery in bacteria that use membrane bound cell division inhibitors.
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Discovery of a two protease DNA Damage Checkpoint recovery mechanism
Cold Spring Harbor Laboratory, 2018Co-Authors: Peter E. Burby, Zackary W. Simmons, Jeremy W. Schroedert, Lyle A. SimmonsAbstract:AbstractThe DNA Damage response is a signaling pathway found throughout biology. In many bacteria the DNA Damage Checkpoint is enforced by inducing expression of a small, membrane bound inhibitor that delays cell division providing time to repair Damaged chromosomes. How cells sense successful DNA repair and promote Checkpoint recovery is unknown. By using a high-throughput, forward genetic screen, we identified two unrelated proteases, YlbL and CtpA, that promote DNA Damage Checkpoint recovery inBacillus subtilis. Deletion of both proteases leads to accumulation of the Checkpoint protein YneA. DNA Damage sensitivity and increased cell elongation in protease mutants depends onyneA. Further, expression of YneA in protease mutants was sufficient to inhibit cell proliferation. Finally, we show that one of the two proteases, CtpA, directly cleaves YneAin vitro. With these results, we report the mechanism for DNA Damage Checkpoint recovery in bacteria that use membrane bound cell division inhibitors.
Randy Yat Choi Poon - One of the best experts on this subject based on the ideXlab platform.
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The CDK1 inhibitory kinase MYT1 in DNA Damage Checkpoint recovery
Oncogene, 2012Co-Authors: Jeremy Pak Hong Chow, Randy Yat Choi PoonAbstract:Inhibition of cyclin-dependent kinase 1 (CDK1) by phosphorylation is a key regulatory mechanism for both the unperturbed cell cycle and the DNA Damage Checkpoint. Although both WEE1 and MYT1 can phosphorylate CDK1, little is known about the contribution of MYT1. We found that in contrast to WEE1, MYT1 was not important for the normal cell cycle or Checkpoint activation. Time-lapse microscopy indicated that MYT1 did, however, have a rate-determining role during Checkpoint recovery. Depletion of MYT1 induced precocious mitotic entry when the Checkpoint was abrogated with inhibitors of either CHK1 or WEE1, indicating that MYT1 contributes to Checkpoint recovery independently of WEE1. The acceleration of Checkpoint recovery in MYT1-depleted cells was due to a lowering of threshold for CDK1 activation. The kinase activity of MYT1 was high during Checkpoint activation and reduced during Checkpoint recovery. Importantly, although depletion of MYT1 alone did not affect long-term cell growth, it potentiated with DNA Damage to inhibit cell growth in clonogenic survival and tumor xenograft models. These results reveal the functions of MYT1 in Checkpoint recovery and highlight the potential of MYT1 as a target for anti-cancer therapies.
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determinants of mitotic catastrophe on abrogation of the g2 DNA Damage Checkpoint by ucn 01
Molecular Cancer Therapeutics, 2011Co-Authors: Kin Fan On, Jeremy Pak Hong Chow, Hoi Tang, Yue Chen, Randy Yat Choi PoonAbstract:Genotoxic stress such as ionizing radiation halts entry into mitosis by activation of the G2 DNA Damage Checkpoint. The CHK1 inhibitor 7-hydroxystaurosporine (UCN-01) can bypass the Checkpoint and induce unscheduled mitosis in irradiated cells. Precisely, how cells behave following Checkpoint abrogation remains to be defined. In this study, we tracked the fates of individual cells after Checkpoint abrogation, focusing in particular on whether they undergo mitotic catastrophe. Surprisingly, while a subset of UCN-01–treated cells were immediately eliminated during the first mitosis after Checkpoint abrogation, about half remained viable and progressed into G1. Both the delay of mitotic entry and the level of mitotic catastrophe were dependent on the dose of radiation. Although the level of mitotic catastrophe was specific for different cell lines, it could be promoted by extending the mitosis. In supporting this idea, weakening of the spindle-assembly Checkpoint, by either depleting MAD2 or overexpressing the MAD2-binding protein p31comet, suppressed mitotic catastrophe. Conversely, delaying of mitotic exit by depleting either p31comet or CDC20 tipped the balance toward mitotic catastrophe. These results underscore the interplay between the level of DNA Damage and the effectiveness of the spindle-assembly Checkpoint in determining whether Checkpoint-abrogated cells are eliminated during mitosis. Mol Cancer Ther; 10(5); 784–94. ©2011 AACR .
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the role of cdc2 feedback loop control in the DNA Damage Checkpoint in mammalian cells
Cancer Research, 1997Co-Authors: Randy Yat Choi Poon, Man Sang Chau, Katsumi Yamashita, Tony HunterAbstract:DNA Damage inactivates cyclin-dependent kinases (CDKs) and arrests the cell cycle. Following DNA Damage, the G1-S CDKs are inhibited by a mechanism involving p53-dependent induction of p21Cip1/Waf1; but how the Cdc2 is inhibited is less apparent. We found that the signal generated by the DNA Damage Checkpoint in G2 was dominant over that from the spindle microtubule-assembly Checkpoint, because the high Cdc2 activity present in nocodazole or Taxol-arrested cells was reduced by DNA Damage. Phosphorylation of the inhibitory residues in Cdc2, Thr14, and Tyr15 coincided with the inactivation of Cdc2 after DNA Damage. Interpretation of this result, however, was not straightforward due to the regulation of Thr14/Tyr15 phosphorylation by feedback loops; hence, their phosphorylation can in principle result merely from the inhibition of Cdc2 activity. Consistent with this, Thr14/Tyr15 phosphorylation was induced when Cdc2 kinase activity was inhibited with butyrolactone-I. Given these complications, we undertook a more critical analysis of the mechanisms that regulate Cdc2 after DNA Damage. Caffeine reversed the DNA Damage-induced inhibition of Cdc2 by causing dephosphorylation of Cdc2, and this dephosphorylation still occurred even when the Cdc2 feedback loops were blocked with butyrolactone-I. These data suggest that the DNA Damage Checkpoint in part acts through Thr14/Tyr15 phosphorylation by a mechanism independent of Cdc2 activity, and this phosphorylation can be accentuated by the Cdc2 feedback loops involving Thr14/Tyr15 protein kinases and phosphatases. The kinase activity of the Wee1Hu Tyr15 protein kinase was unaltered after DNA Damage, but the phosphatase activity of Cdc25C was reduced. Thus, the decrease in Cdc25C activity may in part account for the DNA Damage-induced increase in Thr14/Tyr15 phosphorylation.
Bachar H Hassan - One of the best experts on this subject based on the ideXlab platform.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Rui Ye, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Abstract Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage.
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formation of arabidopsis cryptochrome 2 photobodies in mammalian nuclei application as an optogenetic DNA Damage Checkpoint switch
Journal of Biological Chemistry, 2013Co-Authors: Irem Ozkandagliyan, Yi Ying Chiou, Bachar H Hassan, Nuri Ozturk, Aziz SancarAbstract:Nuclear bodies are discrete suborganelle structures that perform specialized functions in eukaryotic cells. In plant cells, light can induce de novo formation of nuclear bodies called photobodies (PBs) composed of the photosensory pigments, phytochrome (PHY) or cryptochrome (CRY). The mechanisms of formation, the exact compositions, and the functions of plant PBs are not known. Here, we have expressed Arabidopsis CRY2 (AtCRY2) in mammalian cells and analyzed its fate after blue light exposure to understand the requirements for PB formation, the functions of PBs, and their potential use in cell biology. We found that light efficiently induces AtCRY2-PB formation in mammalian cells, indicating that, other than AtCRY2, no plant-specific proteins or nucleic acids are required for AtCRY2-PB formation. Irradiation of AtCRY2 led to its degradation; however, degradation was not dependent upon photobody formation. Furthermore, we found that AtCRY2 photobody formation is associated with light-stimulated interaction with mammalian COP1 E3 ligase. Finally, we demonstrate that by fusing AtCRY2 to the TopBP1 DNA Damage Checkpoint protein, light-induced AtCRY2 PBs can be used to activate DNA Damage signaling pathway in the absence of DNA Damage. Background: AtCRY2 forms photobodies in plants. Results: AtCRY2 forms photobodies in human cells. By fusing AtCRY2 to a human Checkpoint protein, the DNA Damage response can be activated by light. Conclusion: AtCRY2 and its fusion proteins can achieve light-induced protein multimerization independent of other plant proteins. Significance: AtCRY2 can function as an optogenetic tool to modulate signaling pathways.