H2020Индивидуална стипендия2016–2018

DNA Pol III · How single DNA polymerases make decisions during proofreading

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

Период: 2016-04-01 → 2018-06-13
Финансиране от ЕС: 183 455 €
Участници: 1
Схема: MSCA-IF

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Накратко на български

Ензимът ДНК полимераза III се наблюдава в реално време, за да се разбере как открива и поправя грешки при копирането на ДНК. Разбирането на този механизъм помага да се изясни как възникват мутациите и развитието на заболявания като рака.

Този кратък обзор е генериран от изкуствен интелект

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

Резултати накратко

How single DNA polymerases make decisions during proofreading

The main goal of this research was to understand fundamental molecular-level details regarding the mechanism of proofreading with the model bacterial enzyme DNA polymerase III at single-molecule resolution. Proofreading is an important process that occurs during DNA replication to ensure high fidelity. Replicating DNA is a critical aspect of inheritance that is carried out by DNA polymerase enzymes. During DNA replication, DNA polymerases make a new copy of DNA that will be packaged into a new cell. DNA polymerases need to balance replication speed and accuracy. While most DNA polymerases work very fast and with high accuracy, they can make mistakes during replication that can result in a DNA mutation. DNA mutations, if not corrected, can lead to human diseases like cancer. Therefore, understanding mechanisms of proofreading has an important impact on society in order to understand fundamental aspects of mutagenesis and carcinogenesis. This goal of this work is to study how single DNA polymerase enzymes carry out the molecular acrobatic act of proofreading. In order to do this, we have developed a single-molecule system where we can watch single DNA polymerase enzymes perform proofreading in real time. We have monitored the kinetics and protein binding times for DNA polymrease III during the course of a proofreading reaction. We have studied how proofreading changes as a function of the type of error in DNA. Further, we have determined that the conformational dynamics during proofreading for DNA polymerase III change in comparing the wild type and a mutant enzyme that is deficient in proofreading. It is our hope that this work will shift the knowledge frontier by advancing our understanding of proofreading during DNA replication in order to better realise the relationship between DNA mutations and the development of human diseases like cancer.

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

Цел на проекта

DNA polymerases (DNA Pols) make various decisions during DNA replication. Decisions regarding nucleotide selection, lesion bypass and how to respond to replication errors are a few important examples. During replication, DNA Pols can synthesize DNA at remarkable rates, e.g. 1,000 bp/s for E. Coli DNA Pol III. However, errors can be introduced during replication, resulting in a terminal DNA mismatch. Mismatches can then be sensed and removed by action of the 3’ to 5’ exonuclease activity of the DNA Pol. How DNA Pols can sense mismatches to initiate proofreading and how the primer strand migrates into the exonuclease domain, often a distance of 30 Å, is the focus of this proposal. Moreover, this research aims to understand mechanisms of proofreading in higher complexity DNA Pol assemblies at single-molecule resolution. Proofreading will be studied with the model enzyme E. Coli DNA Pol III, consisting of separate protein subunits that assemble to form a higher order complex, namely the polymerase, exonuclease and the β2-clamp, which is a processivity factor that encircles the DNA and increases the affinity of the polymerase to the DNA. The proposed work will employ single-molecule FRET in order to evaluate proofreading dynamics at high spatial and temporal resolution. Three main objectives will be performed in order to understand (1) how DNA mismatches initiate proofreading (2) how mutations within the exonuclease domain affect proofreading and (3) how DNA lesions can influence proofreading as a function of position within the DNA template. These objectives will be achieved with a single-molecule FRET assay containing fluorescently labelled DNA to monitor proofreading dynamics in real time. This work will shift the knowledge frontier by advancing our understanding of proofreading during DNA replication in order to better realise the relationship between DNA mutations and the development of human diseases like cancer.

Оригинален текст от CORDIS (на английски).

Участници

IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE · LondonКоординаторОбединеното кралство