H2020Индивидуална стипендия2019–2021

RecPAIR · Genetic landscape of the homology search

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

Период: 2019-06-08 → 2021-06-07
Финансиране от ЕС: 191 852 €
Участници: 1
Схема: MSCA-IF-EF-ST

Линиите свързват координатора с партньорите.

Накратко на български

Механизмът, по който бактериите E. coli намират подходящ шаблон за поправка на счупената ДНК чрез протеина RecA, се анализира с микроскопия. Разбирането на този процес помага за изясняване на генетични заболявания и рака, тъй като подобни принципи действат и в човешките клетки.

Този кратък обзор е генериран от изкуствен интелект

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

Резултати накратко

Genetic landscape of the homology search

In the RecPAIR project, we focused on the search for the repair template during double-stranded break (DSB) repair via the homologous recombination (HR) pathway using E. coli as a model organism. We used microfluidics and quantitative fluorescence microscopy to unravel the molecular mechanism of the search for the template in live cells. The DSB repair by homologous recombination (HR) is a conserved and abundant mechanism. It deals with a particularly toxic type of DNA damage and serves an important protective role in cell metabolism. Defects in the recombination machinery are linked to genetic diseases and to a high risk of cancer. A detailed description of the molecular mechanism of the HR will help to better understand, and potentially prevent, the pathologies associated with the defects in recombination. DSB repair relies on that the damaged DNA strand can locate the right repair template fast and accurately, but how this is achieved in a living cell has, until now, been misapprehended. The core components and basic mechanistic principles of the DSB repair are shared between even distantly related organisms. The fundamental principles of the recombination mechanism discovered in bacteria likely also apply to eukaryotic cells. The search relies on a prototypic strand exchange protein, RecA in E. coli, which forms a filament of single-stranded DNA (ssDNA) that can locate a homologous target within a double-stranded DNA. In this project, we show that it usually takes a bacterium 10-20 minutes to repair a DSB and that the homology search is completed in less than 9 ± 3 minutes by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose a model in which the architecture of the RecA filament effectively speeds up the search by reducing it from three to two dimensions. Our efforts also resulted in a flexible and universal system to study the process of DSB repair, or other types of DNA damage repair, that can be easily implemented by us, or other researchers in the future. The mentioned system consists of fluorescent fusions of RecA protein, inducible Cas9 nuclease to create DNA damage, microfluidics, and image analysis software.

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

Цел на проекта

The integrity of genetic information is central to life, yet the DNA is vulnerable to damage from internal and external sources. Incorrect repair of DNA damage drives mutagenesis, loss of genetic information, ageing, and cancer. Double strand DNA breaks (DSBs) are perhaps the most threatening DNA lesions, where the integrity of both strands of the DNA duplex is interrupted at the same position. In E. coli, faithful repair of DSBs is possible only through the homologous recombination (HR) pathway which uses replicated chromosome as a template to recover the information. At the center of HR lies an elusive search process, during which broken strand localises and pairs with the repair template.I will use a combination of CRISPR/dCas9 screening and in-situ genotyping of pooled library of strains to characterise the genetic landscape controlling the homology search. First, I will develop a low probability DSB induction method, to limit the DSB-formation to only a single chromosome per cell. Next, I will design and implement a whole-genome CRISPRi screen coupled to high-throughput sequencing and map the genes involved specifically in the homology directed repair of DSBs. The knowledge of the recombination-specific genes will allow to create a refined, high-quality phenotypic screen. In this screen the whole chromosome dynamics will be monitored and defects in the DNA movements will be characterised for each tested target with a microfluidic-based fluorescent microscopy. Each phenotype will be linked to a specific gene using the state-of-the-art in-situ phenotyping approach called DuMPLING. The functional characterisation of recombination genes will allow to conclude a molecular model of the search process in vivo.

Оригинален текст от CORDIS (на английски).

Участници

UPPSALA UNIVERSITET · UppsalaКоординаторШвеция