The present PhD thesis focuses on the structural and the functional analysis of the human DDX11 helicase, a pivotal enzyme for human genome maintenance. DDX11 helicase is a key player in the sister chromatid cohesion process, which ensures the establishment of centromere and the sister chromatid segregation during cell division. Moreover, DDX11 associates to the replisome, ensures faithful DNA replication and participates in DNA repair, unwinding various non-canonical nucleic acids structures. Mutations in DDX11 gene have been associated to the Warsaw Syndrome, a rare cohesinopathy showing phenotypes partially overlapping to Fanconi Anaemia; additionally, DDX11 alterations have been linked to various cancer types. After a bioinformatic analysis of the human DDX11 helicase, we have cloned the full-length DDX11 DNA coding sequence in different expression vectors, harbouring various fusion tags, using the Ligation Independent Cloning technique. DDX11 expression has been tested in a variety of conditions (cell strains and cell lines, temperatures and expression times), testing both bacterial and eukaryotic expression systems. We have successfully optimized a protocol for expression and purification of the full-length protein in baculovirus-infected insect cells. The best expression condition for two different DDX11 constructs has been then scaled up, optimizing a purification protocol and obtaining the protein in amounts suitable for structural studies. Purified DDX11 has been characterized biophysically and biochemically, suggesting the presence of an intact FeS cluster, dissecting its nucleic acids binding properties and testing the helicase activity. DDX11 showed high binding specificity, with a significant affinity for DNA fork substrates, Displacement Loops and unimolecular G-Quadruplexes. Consistent with the binding preference, the protein displayed a strong unwinding activity towards D-loops and a significant activity towards all the DNA-based substrates possessing a 5’ single strand DNA overhang. We have also observed some unwinding activity towards 5’ tail R-loops. DDX11 was reported to be able to unwind bimolecular but not the more physiological unimolecular G-quadruplexes. We used a different assay, that was less prone to some of the limitations of the traditional gel-based assays, and were able to show some preliminary data, suggesting that the protein is able to unwind unimolecular G4, as long as possessing a long 5' tail. With the purified protein, we carried out a large number of crystallization experiments testing diverse crystallization conditions; however, no crystal was obtained. Therefore, the structural analysis of DDX11 was performed using single particle Cryo-Electron Microscopy. Although this technique has shown to be able to tackle smaller and smaller macromolecules, a protein with a size of about 100 kDa or less is still a challenge. After a detailed optimization process of the vitrification conditions, we successfully collected Cryo-EM datasets for DDX11apo-protein and in complex with DNA fork and G-quadruplexes. In the present work, we show the first three-dimensional reconstruction of the human DDX11, reaching a resolution of 5.6 Å. The structural analysis is still very much in progress, and additional Cryo-EM experiments will be performed in the next weeks, improving the results obtained so far.

The present PhD thesis focuses on the structural and the functional analysis of the human DDX11 helicase, a pivotal enzyme for human genome maintenance. DDX11 helicase is a key player in the sister chromatid cohesion process, which ensures the establishment of centromere and the sister chromatid segregation during cell division. Moreover, DDX11 associates to the replisome, ensures faithful DNA replication and participates in DNA repair, unwinding various non-canonical nucleic acids structures. Mutations in DDX11 gene have been associated to the Warsaw Syndrome, a rare cohesinopathy showing phenotypes partially overlapping to Fanconi Anaemia; additionally, DDX11 alterations have been linked to various cancer types. After a bioinformatic analysis of the human DDX11 helicase, we have cloned the full-length DDX11 DNA coding sequence in different expression vectors, harbouring various fusion tags, using the Ligation Independent Cloning technique. DDX11 expression has been tested in a variety of conditions (cell strains and cell lines, temperatures and expression times), testing both bacterial and eukaryotic expression systems. We have successfully optimized a protocol for expression and purification of the full-length protein in baculovirus-infected insect cells. The best expression condition for two different DDX11 constructs has been then scaled up, optimizing a purification protocol and obtaining the protein in amounts suitable for structural studies. Purified DDX11 has been characterized biophysically and biochemically, suggesting the presence of an intact FeS cluster, dissecting its nucleic acids binding properties and testing the helicase activity. DDX11 showed high binding specificity, with a significant affinity for DNA fork substrates, Displacement Loops and unimolecular G-Quadruplexes. Consistent with the binding preference, the protein displayed a strong unwinding activity towards D-loops and a significant activity towards all the DNA-based substrates possessing a 5’ single strand DNA overhang. We have also observed some unwinding activity towards 5’ tail R-loops. DDX11 was reported to be able to unwind bimolecular but not the more physiological unimolecular G-quadruplexes. We used a different assay, that was less prone to some of the limitations of the traditional gel-based assays, and were able to show some preliminary data, suggesting that the protein is able to unwind unimolecular G4, as long as possessing a long 5' tail. With the purified protein, we carried out a large number of crystallization experiments testing diverse crystallization conditions; however, no crystal was obtained. Therefore, the structural analysis of DDX11 was performed using single particle Cryo-Electron Microscopy. Although this technique has shown to be able to tackle smaller and smaller macromolecules, a protein with a size of about 100 kDa or less is still a challenge. After a detailed optimization process of the vitrification conditions, we successfully collected Cryo-EM datasets for DDX11apo-protein and in complex with DNA fork and G-quadruplexes. In the present work, we show the first three-dimensional reconstruction of the human DDX11, reaching a resolution of 5.6 Å. The structural analysis is still very much in progress, and additional Cryo-EM experiments will be performed in the next weeks, improving the results obtained so far.

Structural and Functional Analysis of human DDX11 helicase / Longo, Antonio. - (2024 Feb 27).

Structural and Functional Analysis of human DDX11 helicase

LONGO, ANTONIO
2024-02-27

Abstract

The present PhD thesis focuses on the structural and the functional analysis of the human DDX11 helicase, a pivotal enzyme for human genome maintenance. DDX11 helicase is a key player in the sister chromatid cohesion process, which ensures the establishment of centromere and the sister chromatid segregation during cell division. Moreover, DDX11 associates to the replisome, ensures faithful DNA replication and participates in DNA repair, unwinding various non-canonical nucleic acids structures. Mutations in DDX11 gene have been associated to the Warsaw Syndrome, a rare cohesinopathy showing phenotypes partially overlapping to Fanconi Anaemia; additionally, DDX11 alterations have been linked to various cancer types. After a bioinformatic analysis of the human DDX11 helicase, we have cloned the full-length DDX11 DNA coding sequence in different expression vectors, harbouring various fusion tags, using the Ligation Independent Cloning technique. DDX11 expression has been tested in a variety of conditions (cell strains and cell lines, temperatures and expression times), testing both bacterial and eukaryotic expression systems. We have successfully optimized a protocol for expression and purification of the full-length protein in baculovirus-infected insect cells. The best expression condition for two different DDX11 constructs has been then scaled up, optimizing a purification protocol and obtaining the protein in amounts suitable for structural studies. Purified DDX11 has been characterized biophysically and biochemically, suggesting the presence of an intact FeS cluster, dissecting its nucleic acids binding properties and testing the helicase activity. DDX11 showed high binding specificity, with a significant affinity for DNA fork substrates, Displacement Loops and unimolecular G-Quadruplexes. Consistent with the binding preference, the protein displayed a strong unwinding activity towards D-loops and a significant activity towards all the DNA-based substrates possessing a 5’ single strand DNA overhang. We have also observed some unwinding activity towards 5’ tail R-loops. DDX11 was reported to be able to unwind bimolecular but not the more physiological unimolecular G-quadruplexes. We used a different assay, that was less prone to some of the limitations of the traditional gel-based assays, and were able to show some preliminary data, suggesting that the protein is able to unwind unimolecular G4, as long as possessing a long 5' tail. With the purified protein, we carried out a large number of crystallization experiments testing diverse crystallization conditions; however, no crystal was obtained. Therefore, the structural analysis of DDX11 was performed using single particle Cryo-Electron Microscopy. Although this technique has shown to be able to tackle smaller and smaller macromolecules, a protein with a size of about 100 kDa or less is still a challenge. After a detailed optimization process of the vitrification conditions, we successfully collected Cryo-EM datasets for DDX11apo-protein and in complex with DNA fork and G-quadruplexes. In the present work, we show the first three-dimensional reconstruction of the human DDX11, reaching a resolution of 5.6 Å. The structural analysis is still very much in progress, and additional Cryo-EM experiments will be performed in the next weeks, improving the results obtained so far.
27-feb-2024
36
2022/2023
Settore BIO/10 - Biochimica
Università degli Studi di Trieste
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3071658
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