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How single DNA polymerases make decisions during proofreading

Periodic Reporting for period 1 - DNA Pol III (How single DNA polymerases make decisions during proofreading)

Reporting period: 2016-04-01 to 2018-03-31

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.
The main findings of this work show that DNA polymerase III binds dynamically to DNA during the process of proofreading. Further, the dyanmics change depending on the type of mutation at the end of the DNA terminus. We have further determined the binding rates for DNA polymerase III at various mismatches in DNA. For this work a mutant DNA polymerase III that is deficient in proofreading was tested at a single-molecule level to determine it's kinetics and conformational dynamics in comparison to the wild type enzyme. We found significant changes in the mutant enzyme's dynamics in comparsion to the wild type, which shows that single changes in amino acid content can influence dyanmics during proofreading. The findings of this research were presented at the 2017 Biophysical Society Meeting in New Orleans, USA in March 2017.
This project sucessfully established a single-molecule assay to measure proofreading dynamics for DNA polymerase III. The contribution to the state of the art is the advancement in our understanding of proofreading mechanisms regarding larger protein complexes involved in DNA replication. For this work, the processivity factor, i.e. the beta-clamp in E. coli, was tested for its impact on DNA polymerase III proofreading. To date, less is known regarding proofreading dynamics with DNA polymerases and their processivity factors. This work has highlighted the dynamic nature of DNA polymerase III at a single-molecule level and helps to advance the knowledge frontier in our understanding of proofreading.
Watching DNA polymerase III perform proofreading in real time