Obstacles to replication: uncovering the mechanisms of macromolecular collisions
Lead Research Organisation:
University of York
Department Name: Biology
Abstract
DNA contains information required for life. All organisms must copy their DNA before they can grow or reproduce. Genetic information is copied using a molecular machine, called the replisome, which copies information while travelling along DNA. To prevent corruption of the information in DNA, this duplication process must occur accurately.
When the replisome is travelling along DNA it can be blocked by several types of obstacles. The most common and problematic example is another molecular machine called RNA polymerase. RNA polymerase reads the genes encoded in DNA to help produce molecules required by the cell. In bacteria the replisome and RNA polymerase are both active at the same time, which can lead to collisions on the DNA. Collisions of these molecular machines can damage DNA and this in turn can cause cell death. Furthermore there are other barriers that can also block the replisome. Termination is the final step of DNA replication and occurs when DNA replication machinery travelling in opposite directions meet. In the bacterium Escherichia coli, the Tus protein binds DNA and forms a one-way replication barrier. The replisome can be held at this barrier until termination occurs. In this project we will investigate what happens when the replisome collides with both of these blocks.
Replisome-obstacle collisions can be minimised but not avoided. Cells contain several enzymes called helicases which can unwind DNA and resolve the collisions. Several helicases that do this job have been identified but we do not know exactly how they work. One of our goals is to understand why helicases can resolve some blockages but not others and why their efficiency varies. To do this we will use Tus or RNA polymerase as a replisome stalling tool and then use advanced microscopy to visualise the blocked proteins. Identifying the features of replication machinery blocked at these obstacles will help us to understand what happens at collisions and how they are resolved. We will also determine how long the replication machinery remains assembled once it has collided with an obstacle and measure how close the machinery can get to a RNA polymerase block. Replication obstacles are a problem for all life forms, but we will study these processes in E. coli bacteria since they are easy to manipulate and it represents an excellent model system. Insights from E. coli often have relevance to all life, including humans, because many of our proteins are similar.
Our results will help us understand how DNA is replicated when there are obstacles in the way. Understanding bacterial solutions to this problem will give us insight into the same process in humans. In the long term this work can have an impact on human health research because related helicases have been implicated in cancer predisposition, human disease and ageing. The evolution of resistance to anti-microbial drugs is a huge problem in medicine because antibiotics are used to treat many diseases and also enable safe surgery. The helicases we will investigate are common amongst bacteria and can be essential for their survival. Understanding how they work could help make them new targets for anti-microbial drugs. Antibiotic treatment targeting important processes like DNA replication could improve patient outcomes and reduce the chance of resistance evolving. In addition, it has been shown that chemotherapeutic agents are more efficient when administered alongside drugs that target helicases. The knowledge gained from this project could therefore benefit multiple patient groups in the future. The advanced microscopy we plan to use is an exciting technique in a rapidly expanding field. An immediate benefit is that this project will help reduce the UK skills deficit in this area by training scientists in the specialised techniques and data analysis.
When the replisome is travelling along DNA it can be blocked by several types of obstacles. The most common and problematic example is another molecular machine called RNA polymerase. RNA polymerase reads the genes encoded in DNA to help produce molecules required by the cell. In bacteria the replisome and RNA polymerase are both active at the same time, which can lead to collisions on the DNA. Collisions of these molecular machines can damage DNA and this in turn can cause cell death. Furthermore there are other barriers that can also block the replisome. Termination is the final step of DNA replication and occurs when DNA replication machinery travelling in opposite directions meet. In the bacterium Escherichia coli, the Tus protein binds DNA and forms a one-way replication barrier. The replisome can be held at this barrier until termination occurs. In this project we will investigate what happens when the replisome collides with both of these blocks.
Replisome-obstacle collisions can be minimised but not avoided. Cells contain several enzymes called helicases which can unwind DNA and resolve the collisions. Several helicases that do this job have been identified but we do not know exactly how they work. One of our goals is to understand why helicases can resolve some blockages but not others and why their efficiency varies. To do this we will use Tus or RNA polymerase as a replisome stalling tool and then use advanced microscopy to visualise the blocked proteins. Identifying the features of replication machinery blocked at these obstacles will help us to understand what happens at collisions and how they are resolved. We will also determine how long the replication machinery remains assembled once it has collided with an obstacle and measure how close the machinery can get to a RNA polymerase block. Replication obstacles are a problem for all life forms, but we will study these processes in E. coli bacteria since they are easy to manipulate and it represents an excellent model system. Insights from E. coli often have relevance to all life, including humans, because many of our proteins are similar.
Our results will help us understand how DNA is replicated when there are obstacles in the way. Understanding bacterial solutions to this problem will give us insight into the same process in humans. In the long term this work can have an impact on human health research because related helicases have been implicated in cancer predisposition, human disease and ageing. The evolution of resistance to anti-microbial drugs is a huge problem in medicine because antibiotics are used to treat many diseases and also enable safe surgery. The helicases we will investigate are common amongst bacteria and can be essential for their survival. Understanding how they work could help make them new targets for anti-microbial drugs. Antibiotic treatment targeting important processes like DNA replication could improve patient outcomes and reduce the chance of resistance evolving. In addition, it has been shown that chemotherapeutic agents are more efficient when administered alongside drugs that target helicases. The knowledge gained from this project could therefore benefit multiple patient groups in the future. The advanced microscopy we plan to use is an exciting technique in a rapidly expanding field. An immediate benefit is that this project will help reduce the UK skills deficit in this area by training scientists in the specialised techniques and data analysis.
Technical Summary
DNA replication must be complete and accurate to prevent corruption of genetic information. However, a physiological genomic template has a variety of nucleoprotein barriers that can impede replication fork progression and cause genome instability. Replication and transcription of DNA occur simultaneously in E. coli so RNA polymerase is the most common obstacle encountered by the replisome. The E. coli genome contains a replication fork trap which is mediated by unidirectional Tus-ter barriers that are not detrimental to cell survival. Accessory replicative helicases promote fork movement through nucleoprotein barriers and have been identified in prokaryotes and eukaryotes. Rep is the primary accessory helicase in E. coli. UvrD is a Rep homologue that can partially compensate for the absence of Rep. Both helicases reduce replisome pausing in vivo and in vitro.
Our vision is to develop a full molecular understanding of replisome-obstacle collisions by studying them at the DNA, protein and complex level. We will use Tus and RNAP as our blocks of choice and characterise how collisions occur and how they are resolved by helicases.
We will use electron cryomicroscopy to examine structures of replisome-obstacle complexes and use replication reconstitution to biochemically analyse replication products and assess the determinants of accessory helicase function. We will characterise a replicative helicase-Tus collision interface with and without Rep present to understand helicase accessibility and Rep function. We will compare this to a minimal replisome-RNAP structure while also measuring replisome progression when blocked by RNAP. Site-directed mutagenesis will enable us to probe potential mechanisms and understand how accessory helicases resolve replication-transcription conflicts in both orientations. This project will improve our understanding of the fundamental process of DNA replication and how accessory helicases enable duplication of physiological templates.
Our vision is to develop a full molecular understanding of replisome-obstacle collisions by studying them at the DNA, protein and complex level. We will use Tus and RNAP as our blocks of choice and characterise how collisions occur and how they are resolved by helicases.
We will use electron cryomicroscopy to examine structures of replisome-obstacle complexes and use replication reconstitution to biochemically analyse replication products and assess the determinants of accessory helicase function. We will characterise a replicative helicase-Tus collision interface with and without Rep present to understand helicase accessibility and Rep function. We will compare this to a minimal replisome-RNAP structure while also measuring replisome progression when blocked by RNAP. Site-directed mutagenesis will enable us to probe potential mechanisms and understand how accessory helicases resolve replication-transcription conflicts in both orientations. This project will improve our understanding of the fundamental process of DNA replication and how accessory helicases enable duplication of physiological templates.
