Pushing proteins off DNA - how do helicases unwind protein-coated DNA?

DNA encodes all the genetic information needed to act as a blueprint for life. This blueprint, though, needs to be converted ultimately into the building blocks of a cell. However, the information that DNA stores is buried deep within the structure of this molecule and can only be accessed by stripping apart the two strands that constitute DNA, so-called unwinding. This critical function is performed by enzymes called helicases that are tiny nanomotors that ratchet along DNA, separating the two DNA strands as they go. All organisms have a variety of different helicases and defects in just one type of helicase can result in catastrophe for a cell, potentially causing lethality or debilitating mutations within genes.

The central importance of helicases in maintaining the viability of all cells and in passing the genetic blueprint from one generation to the next has been known for decades. Recently we have also come to appreciate that helicases face a particular problem when attempting to unwind DNA. DNA is coated in a wide variety of different proteins inside cells. These proteins play important roles in packaging DNA, reading the genetic blueprint and coordinating the movement of chromosomes inside cells. Unfortunately we now know that these bound proteins also present problems to helicases since any proteins bound to the DNA must also be pushed off to allow separation of the two DNA strands by a helicase. However, we know very little about how helicases displace proteins bound to the nucleic acid.

We have been studying a helicase called Rep that plays an important role in copying of the genetic blueprint by displacing proteins that are bound to DNA. Our preliminary work has discovered that removal of part of this helicase activates DNA unwinding but at the same time inhibits displacement of proteins from the DNA. This discovery is important because it shows that displacement of proteins from DNA must involve something more than the helicase merely ratcheting along the DNA. This helicase has therefore evolved specific features to help push proteins off DNA although currently we do not understand what these features are. We aim to investigate how this helicase displaces proteins from DNA by using a combination of different molecular tools to investigate the properties of Rep and versions of this helicase that have increased or decreased abilities to push proteins off DNA. This work will cast light on how this important class of enzyme deals with the vast array of proteins that coat DNA. This problem is one that all organisms must face and so our findings will help us to understand how DNA is maintained effectively inside cells and, just as importantly, how things might go wrong. Mistakes made by helicases can result in very harmful rearrangements within the genetic code, contributing to genetic disease, and so our proposed work will shed light on potential sources of corruption of the genetic code. Conversely, this work may also reveal new ways of deliberately inhibiting helicases. Such inhibitors have potential uses as antiviral, antibacterial and anticancer compounds since helicases are so important for survival.

A central feature of nucleic acid metabolism is the unwinding and remodelling of DNA and RNA via the action of helicases. We know much about how these motor enzymes couple ATP hydrolysis to translocation along and disruption of base pairing between nucleic acid strands. However, recent work has highlighted the barriers to unwinding presented by the many different proteins unavoidably bound to DNA and RNA inside cells. Helicases must therefore disrupt the many noncovalent bonds between nucleic acids and bound proteins in addition to the unwinding of base pairs. A widely held assumption is that protein displacement is simply a consequence of helicase translocation along the nucleic acid. However, in spite of the importance of disrupting nucleoprotein complexes, we know little about how helicases catalyse displacement of proteins from nucleic acids.

The E. coli helicase Rep catalyses protein displacement ahead of the advancing DNA replication machinery. We have created versions of Rep that display (1) activated DNA unwinding but reduced protein displacement, (2) barely detectable DNA unwinding but robust protein displacement and (3) activation of both DNA unwinding and protein displacement. These data demonstrate that nucleoprotein complex disruption is not simply a consequence of helicase translocation along DNA but is an evolved function of these molecular motors. We will exploit these Rep mutants to determine the properties of Rep that are needed to displace proteins from DNA, test whether helicases can act as molecular springs to facilitate disruption of nucleoprotein complexes and analyse the links between helicase properties, protein displacement and force generation. These analyses will provide fundamental mechanistic insight into how molecular motors push proteins off DNA, a key function that sits at the heart of nucleic acid metabolism in all organisms.

This proposal seeks to understand how a class of enzymes central to all aspects of nucleic acid metabolism, helicases, act on their true substrates. All nucleic acids in cells are coated with a vast array of different proteins and so helicases must disrupt protein-nucleic acid interactions in order to unwind the nucleic acid. This ability to disrupt nucleoprotein complexes is therefore critical for the survival of all cells and for the ability to generate faithful copies of their genetic material to pass on to the next generation.

Understanding the mechanistic features of helicases that are critical in disruption of nucleoprotein complexes will further our understanding of potential defects in nucleic acid metabolism and how these might relate to disease. Defects in DNA replication, transcription, translation and recombination are associated with a range of human and animal genetic diseases. Clinicians and scientists with interests in nucleic acid metabolism and links with disease will therefore benefit from the fundamental studies proposed here on how nucleic acids are unwound and remodelled within the complex environment of cells.

A second set of beneficiaries will be scientists and clinicians who wish to develop antiviral, antibacterial and anticancer agents. Helicases have been the targets of many screens for novel inhibitors, in part because this class of enzymes play central roles in maintaining viability. Our proposed work will build on our preliminary data indicating that helicases have evolved specific mechanistic features that aid protein displacement. This proposal will therefore link helicase structure with novel mechanistic features, fundamental information that will be of long-term benefit to pharmaceutical companies aiming to develop new therapies to prevent growth of viruses, bacteria and cancer cells.

Understanding how helicases function will also benefit the biotechnology sector. Development of synthetic organisms requires robust nucleic acid metabolic systems. Central to such systems are helicases and our preliminary work shown in this proposal demonstrates that a core evolved function of helicases is to disrupt nucleoprotein complexes effectively. Our proposed studies will therefore inform the design of nucleic acid metabolic networks needed for synthetic organisms. Helicases are also under development as molecular motors for new very high throughput DNA and RNA sequencing methods using nanopores. Our proposed work will potentially benefit optimisation of these motors for use in sequencing and other related technologies.

This proposal will develop novel applications for cutting-edge biophotonics instrumentation using bespoke equipment that is not available commercially. Direct visualisation of events as they happen at the single molecule level has transformed our understanding of biological systems and consequently there is a growing interest in these advanced imaging techniques. Our proposed experiments will highlight novel applications of these technologies and may aid the development and marketing of commercial biophotonics instrumentation.

This proposed project is highly interdisciplinary. The staff on this project will receive excellent cross-disciplinary training that will add considerably to the scientific skills base within the UK. The UK economy will benefit from this training by enhanced competitiveness in technology-driven industries. Understanding how key enzymes of nucleic acid metabolism function, and what can go wrong with these enzymes, will also have longer term benefits to the health and well-being of the UK population.