H2020Индивидуална стипендия2022–2024

DNA-Rep-EM · Origin-dependent DNA replication visualised by Cryo-EM

„Хоризонт 2020“ — Действия „Мария Склодовска-Кюри“

Период: 2022-09-01 → 2024-08-31
Финансиране от ЕС: 224 934 €
Участници: 1
Схема: MSCA-IF

Линиите свързват координатора с партньорите.

Накратко на български

Репликацията на ДНК при еукариотите се изследва чрез визуализиране на протеиновите комплекси, които копират генетичната информация. Разбирането на тези механизми помага да се разберат причините за генетични заболявания и рак, причинени от грешки при копирането на генома.

Този кратък обзор е генериран от изкуствен интелект

Кратко обяснение, генерирано от езиков модел по текста на CORDIS. Оригиналът е по-долу.

Резултати накратко

Origin-dependent DNA replication visualised by Cryo-EM

The accurate transfer of genetic information from parental to daughter cells requires that the genome be faithfully copied only once per cell cycle. Failures at any point in this process can lead to cellular abnormalities, genetic disease, and cancer. DNA replication is catalysed by the replisome, a large multi-protein complex that coordinates DNA unwinding and synthesis. A hierarchy of strong and weak functional pair-wise interactions controls and maintains the replisome allowing it to transition through multiple conformational states. These transitions are intimately linked to a small number of essentially irreversible chemical steps (ATP-hydrolysis, RNA-primer and DNA synthesis), which determine reaction directionality. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. This project aims to visualise origin-dependent eukaryotic DNA replication using in vitro reconstituted cellular reactions in their entirety, at near atomic resolution. These data will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication. This project has shed some valuable light on the previously unknown mechanism of origin DNA unwinding and spatial orientation of key replisome components.

Текст от CORDIS, на английски · Данни: CORDIS, © Европейски съюз

Цел на проекта

Genome duplication is essential for cell proliferation. Errors in the mechanisms that control DNA replication can cause genomic instability and lead to the development of genetic diseases and cancer. In vitro reconstitution of DNA replication with purified yeast proteins has helped uncover important mechanisms of DNA replication. How changes in protein structure regulates function during key events in origin activation and replisome progression, such as melting of the DNA duplex upon CMG helicase assembly, or the mode of binding of Pol alpha during primer synthesis remain unknown. To date, structural studies have focused on imaging artificially isolated replication complexes using simplified DNA substrates to understand DNA unwinding and replisome architecture. To truly understand the mechanisms that control DNA replication, future structural studies must not only visualise isolated complexes, but also reconstituted reactions. To address this issue, I will integrate single-particle cryo electron-microscopy (cryo-EM) with sophisticated biochemical approaches to image origin-dependent DNA replication reactions in vitro on native DNA substrates. To do so, I will establish short origin-containing DNA substrates that permit loading of a single bidirectional replication fork, allowing large numbers of protein bound origins to be visualised within a single field of view. Initially, I will investigate how the structure of duplex DNA changes upon CMG formation during origin activation. Next, I will examine the molecular mechanisms of primer synthesis after origin activation using both cryo-EM and in vitro DNA replication reactions. Finally, I will capture and image synthesising intact replisomes at near atomic resolution. By visualising entire DNA replication reactions instead of isolated replication complexes at high resolution, we will gain a deeper understanding of the molecular mechanisms that permit the eukaryotic replisome to function during genome duplication.

Оригинален текст от CORDIS (на английски).

Участници

THE FRANCIS CRICK INSTITUTE LIMITED · LondonКоординаторОбединеното кралство