Replication Recovery Through Recombination and Mus81-Mms4
Initial Award Abstract
During DNA synthesis, cells are very vulnerable to agents which damage the DNA template because they inhibit the ability of replication proteins to unwind the double helix or add the next DNA base. This can lead to a total disruption of replication in a region of the genome, but fortunately cells have pathways to restart replication. Smokers put these pathways to the test by exposing themselves to the carcinogens in tobacco products, which reek havoc by creating chemical DNA additions which block normal replication. Within the last decade, a protein complex has been discovered, called Mus81-Mms4 (Mus81 for short), which has been implicated in protecting cells from agents which interfere with DNA replication. Mus81 is a structure-selective endonuclease, which is a protein that cuts DNA strands in structures it recognizes based on the three-dimensional contours of the branched DNA molecules it binds to. It is now believed that at least one function for Mus81 is to create a break at a site where DNA replication has reached a catastrophe. This incision by Mus81 generates a DNA break where a partially replicated chromosome is separated from the chromosome that was being replicated. This DNA break can find the sequence where it left off and reinitiate a replication fork at the same or very similar DNA sequence in the genome with the help of the recombination proteins. Without this measure, the cell would lose some of its genes or have them rearranged so that they do not function properly to protect cells from cancer.
In this project we have two main goals. The first is to test whether two major carcinogenic compounds found in tobacco products create damage for which the Mus81 gene is required for resistance. To do this, yeast strains will be created which lack a functional Mus81 gene and be made subject to tobacco carcinogens. Loss of viability in the mus81 mutants compared with normal cells would indicate the Mus81 gene is required for making an incision in response to these compounds interfering with DNA replication. We can also look at the effects of removing other genes in the same and other pathways supporting replication to get a picture of how Mus81 fits in with the complex network of proteins interacting with the DNA at replication forks. The yeast system is the best model to dissect this complexity because of its advanced genetic capabilities.
The second part of the project is to test the hypothesis that Mus81 cuts a special type of DNA molecule that is formed by recombination proteins. Recombination involves the pairing of homologous DNA strands together, resulting in a ‘joint molecule’, one structure arising from multiple DNA molecules. It is not known whether these structures form before Mus81 delivers the strand incision that leads to the DNA break, but several lines of evidence suggest this may be the case. By reconstituting reactions with Mus81 and key recombination proteins we hope to be able to directly test our hypothesis that Mus81 acts on recombination intermediates that arise after a replication fork is stalled.
Knowledge of the type of DNA structure that Mus81 cuts will help us understand when Mus81 will act and what has to happen first. Most chemotherapeutic agents are actually replication poisons that preferentially kill cancer cells because these cells are growing faster than normal tissue. Since Mus81’s function is critical for the support of challenged replication, it directly opposes the action of these therapeutics trying to kill the replicating cancer cells. For this reason, Mus81 might prove to be a very specific target for sensitizing replicating cells (cancer cells) to chemotherapeutics. Our work will significantly enhance our understanding of the cellular response to the high levels of DNA lesions that are formed from smoking tobacco and how this can lead to lung cancer. It will also potentially identify Mus81 as a candidate tumor-suppressor gene for tobacco smoke-induced cancers. |