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Article

Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?

1
Department of Medical Biotechnology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30387 Kraków, Poland
2
Department of General Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 30387 Kraków, Poland
*
Author to whom correspondence should be addressed.
Equally contributed as first authors.
Antioxidants 2021, 10(1), 94; https://doi.org/10.3390/antiox10010094
Received: 9 November 2020 / Revised: 23 December 2020 / Accepted: 7 January 2021 / Published: 12 January 2021
(This article belongs to the Special Issue Pharmacological and Clinical Significance of Heme Oxygenase-1)
G-quadruplexes (G4) are stacked nucleic acid structures that are stabilized by heme. In cells, they affect DNA replication and gene transcription. They are unwound by several helicases but the composition of the repair complex and its heme sensitivity are unclear. We found that the accumulation of G-quadruplexes is affected by heme oxygenase-1 (Hmox1) expression, but in a cell-type-specific manner: hematopoietic stem cells (HSCs) from Hmox1−/− mice have upregulated expressions of G4-unwinding helicases (e.g., Brip1, Pif1) and show weaker staining for G-quadruplexes, whereas Hmox1-deficient murine induced pluripotent stem cells (iPSCs), despite the upregulation of helicases, have more G-quadruplexes, especially after exposure to exogenous heme. Using iPSCs expressing only nuclear or only cytoplasmic forms of Hmox1, we found that nuclear localization promotes G4 removal. We demonstrated that the proximity ligation assay (PLA) can detect cellular co-localization of G-quadruplexes with helicases, as well as with HMOX1, suggesting the potential role of HMOX1 in G4 modifications. However, this colocalization does not mean a direct interaction was detectable using the immunoprecipitation assay. Therefore, we concluded that HMOX1 influences G4 accumulation, but rather as one of the proteins regulating the heme availability, not as a rate-limiting factor. It is noteworthy that cellular G4–protein colocalizations can be quantitatively analyzed using PLA, even in rare cells. View Full-Text
Keywords: heme oxygenase-1; heme; G-quadruplex; proximity ligation assay heme oxygenase-1; heme; G-quadruplex; proximity ligation assay
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MDPI and ACS Style

Krzeptowski, W.; Chudy, P.; Sokołowski, G.; Żukowska, M.; Kusienicka, A.; Seretny, A.; Kalita, A.; Czmoczek, A.; Gubała, J.; Baran, S.; Klóska, D.; Jeż, M.; Stępniewski, J.; Szade, K.; Szade, A.; Grochot-Przęczek, A.; Józkowicz, A.; Nowak, W.N. Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes? Antioxidants 2021, 10, 94. https://doi.org/10.3390/antiox10010094

AMA Style

Krzeptowski W, Chudy P, Sokołowski G, Żukowska M, Kusienicka A, Seretny A, Kalita A, Czmoczek A, Gubała J, Baran S, Klóska D, Jeż M, Stępniewski J, Szade K, Szade A, Grochot-Przęczek A, Józkowicz A, Nowak WN. Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes? Antioxidants. 2021; 10(1):94. https://doi.org/10.3390/antiox10010094

Chicago/Turabian Style

Krzeptowski, Wojciech, Patryk Chudy, Grzegorz Sokołowski, Monika Żukowska, Anna Kusienicka, Agnieszka Seretny, Agata Kalita, Alicja Czmoczek, Jakub Gubała, Sonia Baran, Damian Klóska, Mateusz Jeż, Jacek Stępniewski, Krzysztof Szade, Agata Szade, Anna Grochot-Przęczek, Alicja Józkowicz, and Witold N. Nowak. 2021. "Proximity Ligation Assay Detection of Protein–DNA Interactions—Is There a Link between Heme Oxygenase-1 and G-quadruplexes?" Antioxidants 10, no. 1: 94. https://doi.org/10.3390/antiox10010094

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