Preventing too much of a good thing - conferring DNA structure specificity on a replicative helicase via its loader.

Lead Research Organisation: University of Aberdeen
Department Name: School of Medical Sciences


A major problem faced by all life forms is how to copy the genetic material so it can be passed on to the next generation. This copying of DNA must be done with the highest possible accuracy so that potentially disastrous errors in the genetic code are minimised. The magnitude of this challenge is enormous, given the huge amounts of DNA that constitute the genetic blueprint of even simple organisms such as bacteria. As a result, all organisms have evolved complex protein machines that copy DNA both with high accuracy and rapidity. Moreover, the initiation of DNA replication is a tightly controlled process in all types of organism, ensuring that replication occurs only when and where required. The importance of controlling replication initiation is highlighted by the genome instability that may arise as a result of uncontrolled chromosome duplication. A central feature of DNA replication is the motor that drives the replication machinery forward and separates the two DNA strands prior to their being copied. This motor in the bacterium E. coli is DnaB helicase, and the key event in initiation of DNA replication is the loading of DnaB onto the chromosome. DnaB binds to just one DNA strand during DNA replication, moving along this strand as it separates the double stranded DNA ahead of it. However, DnaB can move along double stranded as well as single stranded DNA. It has been suggested that this surprising property allows DnaB, and possibly replicative helicases in other organisms, to participate in reactions that are used to rearrange genomes rather than to copy genomes with the highest possible accuracy. However, such a role for DnaB appears to contradict the requirements for controlled initiation and accurate replication of chromosomes. Our preliminary data has shown that this paradox might be resolved via a protein that helps to load DnaB onto single stranded DNA. This protein, DnaC, may confer DNA structure specificity on DnaB thereby preventing DnaB from sliding along double stranded DNA. We aim to investigate the mechanism of this DNA structure specificity, the potential roles of DnaC-directed DNA structure selectivity on DnaB function in vivo, and establish whether these properties of the E. coli replicative helicase and its loader are general features of the bacterial replication machinery. Furthermore, our data indicate that DnaC and DnaG, another essential enzyme that interacts with the helicase, may compete for binding to DnaB. We aim to establish whether DnaC impacts upon the function of DnaG during replication, and whether the balance between C and G plays a role in regulation of genome duplication. These studies will establish whether movement of a replicative helicase along double stranded DNA is a pathological event which has necessitated the evolution of mechanisms to avoid such a reaction, rather than a reaction which has been harnessed by the cell to promote genome rearrangements. They may also illuminate how interactions between the motor that drives replication and other essential replication enzymes have evolved to facilitate rapid, accurate chromosome duplication.

Technical Summary

Ring helicases catalyse separation of the two parental DNA strands ahead of replication forks and also interact with other essential replisome components. Loading of the replicative helicase onto ssDNA is the key event in replication fork assembly. This assembly process is tightly controlled as replication initiation at the wrong time and/or in the wrong place on the chromosome would lead to genome instability. In this context, recent findings that these ring helicases can move along double stranded as well as ssDNA were surprising, pointing to a possible role for such helicases in other aspects of DNA metabolism such as recombination. This DNA substrate promiscuity appears to conflict with the requirement for tight regulation of replication. Our recent data suggests that this paradox is resolved in E. coli by the replicative helicase loader DnaC. Previous work suggested that DnaC acted only to load DnaB onto single stranded DNA, playing no part in subsequent DnaB catalysis. Our current work indicates that DnaC may function in concert with DnaB at all stages of DnaB catalysis, acting to ensure that DnaB translocates only along single stranded and not dsDNA. Moreover, DnaC may influence primase activity during DNA replication via DnaB, thus regulating lagging strand synthesis. This proposal aims to determine the mechanism governing this DnaC-dependent DNA structure specificity and to investigate the potential function of DnaC in regulating DnaB and DnaG catalysis. We will also investigate whether other bacterial replicative helicases share the DNA structure specificity of E. coli DnaB and C highlighted by our own work. These studies will establish whether restriction of replicative helicase catalysis is a requirement for DNA replication imposed by the observed substrate promiscuity of ring helicases, an apparently ubiquitous feature of such helicases throughout all domains of life.


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Description Copying the genetic material is a key process in all organisms since when a cell divides it must pass on a complete, very accurate copy of all its genetic information to both daughter cells. Central to this copying process is the un-zipping of the two strands within the DNA double helix to allow enzymes to copy the bases (the "text" of the genetic code) within the DNA. This unzipping of the DNA strands is performed by an enzyme called a replicative helicase. This enzyme binds to just one of the two strands within the DNA double helix and, by moving along this one strand, the helicase acts as a "wire stripper" to unzip the two DNA strands. However, several studies have indicated that the replicative helicase can move along not only one strand of DNA but also along double-stranded DNA. This movement along the double-stranded DNA might be involved in reactions that cause rearrangements within the genetic material rather than copy genomes with the highest possible accuracy. This study aimed to establish whether cells contain mechanisms to prevent replicative helicases catalysing rearrangements within the genetic material, potentially harmful events that could cause mutations. The bacterium E. coli was used as a model to analyse these processes, exploiting the powerful experimental tools available in this organism.The key findings from this project were:1) a protein called DnaC which is known to interact with the replicative helicase in E. coli can alter the specificity of the helicase (DnaB) when moving along DNA, preventing the helicase from moving along double-stranded DNA. These data indicate that specific mechanisms exist to prevent the replicative helicase from promoting mutations within the genetic material.2) the DnaC protein prevents DnaB from moving along double-stranded DNA by "sensing" the structure of the DNA in front of the helicase. We also discovered the nature of the molecular switch within DnaC that allows this protein to sense the structure of the DNA.3) a third protein known to interact with the replicative helicase does not impact directly on the regulation of DnaB activity by DnaC. This finding, whilst negative, resulted in us discovering a novel interaction between DnaB and another protein
Exploitation Route This work has uncovered how one of the key enzymes that catalyse DNA replication is regulated so as to prevent unrestricted activity. This unrestricted activity might promote the formation of mutations within the genome of an organism. Our work may lead to the development of a better understanding of how mutations are caused inside all organisms, given the high degree of conservation within the DNA replication machinery across all forms of life.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

Description The nature of this type of research is long-term and it is too early to expect our findings to be exploited by others.
First Year Of Impact 2014