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Cryo-EM studies of Phosphatidylinositol-3-kinase related kinase (PIKK) structures from yeast: Transcription-associated protein 1 (Tra1) and Mitosis-entry checkpoint 1 (Mec1), and their role as conserved genomic regulators

Díaz-Santín, Luis Miguel; (2021) Cryo-EM studies of Phosphatidylinositol-3-kinase related kinase (PIKK) structures from yeast: Transcription-associated protein 1 (Tra1) and Mitosis-entry checkpoint 1 (Mec1), and their role as conserved genomic regulators. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The regulation of the genome is an essential biological task. In eukaryotes, fundamental nuclear processes such as DNA replication, transcription and repair require a large variety of protein effectors to ensure the correct transmission of genetic information, for regulated gene expression and to maintain genome integrity. The phosphatidylinositol 3-kinase-related kinase (PIKK) protein family represents a group of very large and highly conserved Ser/Thr-protein kinases, where most function in genome regulation processes. S. cerevisiae proteins Tra1 and Mec1 (known as TRRAP and ATR in humans) are two essential PIKKs found in the nucleus. Tra1 is a 3,744-residue protein and a shared subunit between the chromatin modifying complexes SAGA and NuA4. Tra1 stimulates transcriptional activation by directly interacting with transcription activators and is also involved in DNA repair. Here, I present the near-atomic resolution cryo-EM structure of the full-length Tra1 polypeptide. A “de novo” atomic model was built from the map, revealing the unique arrangement of Tra1 alpha-solenoid, which is organised into an N-terminal Finger segment inserted into a circular Cradle region before the Kinase domain. Structural interpretation of previously published Tra1 mutants allowed to identify a Gal4 binding region. Mec1 is a 2,368-residue protein that forms a complex with Ddc2 (ATRIP in humans), and participates in the DNA-Damage-Response, Replication-Stress-Response, in telomere maintenance and meiosis, and its kinase activity influences cell cycle progression. Here, I present the high-resolution cryo-EM structure of the heterodimeric Mec1-Ddc2 complex, providing the first complete atomic model of Mec1. The structure reveals how the complex is assembled, showing extensive interactions between Ddc2 solenoid region and Mec1. The map also shows the integration point of Ddc2 N-terminal region into the complex, and together with existing structural data for the interaction of Ddc2 coiled-coil with RPA, an integrative model shed light in the understanding of Mec1 recruitment to RPA-coated ssDNA.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: Cryo-EM studies of Phosphatidylinositol-3-kinase related kinase (PIKK) structures from yeast: Transcription-associated protein 1 (Tra1) and Mitosis-entry checkpoint 1 (Mec1), and their role as conserved genomic regulators
Event: UCL (University College London)
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Copyright © The Author 2021. Original content in this thesis is licensed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0) Licence (https://creativecommons.org/licenses/by-nc/4.0/). Any third-party copyright material present remains the property of its respective owner(s) and is licensed under its existing terms. Access may initially be restricted at the author’s request.
UCL classification: UCL
UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences
UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences
URI: https://discovery.ucl.ac.uk/id/eprint/10122089
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