Analysis of MCM2-7 and MCM8 protein functions and interactions in Drosophila melanogaster.
Lead Research Organisation:
St George's, University of London
Department Name: Basic Medical Sciences
Abstract
When cells divide each new cell needs to have an accurate copy of the genomic DNA if it is to survive. The process by which this occurs is DNA replication. When this process goes wrong the consequences for the cell are bad. New cells may receive partial genomes, a situation which is usually lethal. However in some cases the effects are more subtle. This often leads to the production of cells with mutations which may function poorly. Another effect can be the production of unstable genomes and this situation is often the first stage in the production of cancers.
The work we are proposing to carry out is directed towards an understanding of one part of the protein machinery involved in the process of DNA replication. The proteins we will study are vital cogs in the replication machinery without which the process cannot take place. If we can understand in detail the way that these proteins function we will understand DNA replication better. This will lead to a better understanding of the ways that cells work. More importantly in the long term it may also lead to an understanding of human disease. Some of the proteins that we are looking at have been seen to be altered in cancers. They have also been used as the basis for new screening techniques for cervical and other types of cancers. We therefore hope that our work will ultimately lead to a better understanding of the ways that diseases occur when things go wrong with a basic cellular process such as DNA replication and the development of new ways to diagnose and treat those diseases.
The work we are proposing to carry out is directed towards an understanding of one part of the protein machinery involved in the process of DNA replication. The proteins we will study are vital cogs in the replication machinery without which the process cannot take place. If we can understand in detail the way that these proteins function we will understand DNA replication better. This will lead to a better understanding of the ways that cells work. More importantly in the long term it may also lead to an understanding of human disease. Some of the proteins that we are looking at have been seen to be altered in cancers. They have also been used as the basis for new screening techniques for cervical and other types of cancers. We therefore hope that our work will ultimately lead to a better understanding of the ways that diseases occur when things go wrong with a basic cellular process such as DNA replication and the development of new ways to diagnose and treat those diseases.
Technical Summary
The MCM2-7 proteins are six related proteins which interact with each other and function in DNA replication in eukaryotic cells. They are thought to act at both the initiation stage of replication during the formation of the preRC complex and during elongation as a helicase. MCM8 is a recently identified relative of MCM2-7 which also has helicase activity and has been implicated in initiation and elongation. The research proposed here will look at three areas related to this group of proteins.
? The ?MCM paradox? is the observation that although the MCM2-7 proteins are very abundant, not all of this protein content is needed for replication in higher eukaryotes. A recent study in vitro suggested that the extra MCM content is needed for the cell to respond to replication stress. We have preliminary results that suggest that this may also be true in vivo. However our results also suggest that the in vivo and in vitro situations are not entirely analogous. We therefore propose to investigate the mechanisms involved in the requirement for high levels of MCM proteins in the response to replication stress and DNA damage in vivo. We will also investigate whether this is the only reason for the excess of MCM proteins.
? The cellular function of MCM8 is not fully understood. We will therefore investigate the role of MCM8 in S2 cells and look at its interaction with MCM2-7.
? Finally it is not known whether other cellular helicases contribute to DNA replication. We will therefore investigate functional interactions of other helicases with the MCM proteins as a first step to determining whether they have a replication role.
These studies will be carried out using Drosophila S2 cells. The similarities between Drosophila and other higher eukaryotes mean that the results obtained here could be easily extrapolated into other systems. However Drosophila cells are more readily amenable to RNAi and stable cell line production. These two techniques are central to this proposal where they will be used for the targeted depletion of specific proteins. Cell biological and biochemical approaches such as immuno-fluoresence, FACS analysis, cellular fractionation and immuno-precipitation will then be used to determine the cellular effects of these depletions.
These studies will make a significant contribution to our understanding of DNA replication and therefore of cell function. In the long term the information gained may also find application in the understanding, diagnosis and treatment of disease.
? The ?MCM paradox? is the observation that although the MCM2-7 proteins are very abundant, not all of this protein content is needed for replication in higher eukaryotes. A recent study in vitro suggested that the extra MCM content is needed for the cell to respond to replication stress. We have preliminary results that suggest that this may also be true in vivo. However our results also suggest that the in vivo and in vitro situations are not entirely analogous. We therefore propose to investigate the mechanisms involved in the requirement for high levels of MCM proteins in the response to replication stress and DNA damage in vivo. We will also investigate whether this is the only reason for the excess of MCM proteins.
? The cellular function of MCM8 is not fully understood. We will therefore investigate the role of MCM8 in S2 cells and look at its interaction with MCM2-7.
? Finally it is not known whether other cellular helicases contribute to DNA replication. We will therefore investigate functional interactions of other helicases with the MCM proteins as a first step to determining whether they have a replication role.
These studies will be carried out using Drosophila S2 cells. The similarities between Drosophila and other higher eukaryotes mean that the results obtained here could be easily extrapolated into other systems. However Drosophila cells are more readily amenable to RNAi and stable cell line production. These two techniques are central to this proposal where they will be used for the targeted depletion of specific proteins. Cell biological and biochemical approaches such as immuno-fluoresence, FACS analysis, cellular fractionation and immuno-precipitation will then be used to determine the cellular effects of these depletions.
These studies will make a significant contribution to our understanding of DNA replication and therefore of cell function. In the long term the information gained may also find application in the understanding, diagnosis and treatment of disease.
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| Sue Cotterill (Principal Investigator) |