The major objective of the work done in our laboratory is to understand the processes by which normal cells become cancerous. Since genome instability is a hallmark of the cancer phenotype as well as most likely being a contributor to tumorigenesis, our research specifically focuses on defining how genome integrity is maintained.
More specifically the current focus of our research program is to understand how cells protect the integrity of their genome and how they deal with DNA damage.
We are using genetic, biochemical, functional genomic and proteomic approaches as well as collaborating with structural biologists, computational biologists and mouse geneticists to achieve these goals.
Our work not only finds applications in cancer biology but also in the biology of aging, evolution and stem cell maintenance. We currently mainly employ mammalian cells to carry out these studies but we have a thriving group within the lab that employs the awesome power of the genetics of budding yeast to carry out these studies. We are also initiating the genetic dissection of genome instability pathways in mouse and this will likely be a growth area in the next few years.
We have a wide range of projects that can be broadly grouped in the following three sub-projects:
Orthwein A, Fradet-Turcotte A, Noordermeer SM, Canny MD, Brun CM, Strecker J, Escribano-Diaz C, Durocher D.
Mitosis Inhibits DNA Double-Strand Break Repair to Guard Against Telomere Fusions.
Science. 2014 Mar 20. PMID: 24652939
When DNA is damaged, cells not only attempt to rapidly repair the lesion but almost invariably trigger a signaling cascade that alters many aspects of the cell’s physiology. We are interested in how this signaling cascade is initiated but also, importantly, how it is maintained and terminated.
In the past few years, we have discovered that regulatory ubiquitylation plays a critical role in the orchestration of the response to DNA double-strand breaks. Indeed, using genome-scale RNA interference screening coupled to high-throughput microscopy, we have discovered the RNF8 and RNF168 E3 ubiquitin ligases (Kolas et al. 2007 Science; Stewart et al. Cell 2009). They both cooperate with the UBC13 E2 conjugating enzyme to build lysine 63-(K63) ubiquitin chains at the site of DNA damage. These chains then play a key role in the recruitment of the DNA damage repair and signaling proteins 53BP1 and BRCA1 (for a review: Panier & Durocher. 2009 DNA repair). The clinical importance of this pathway was demonstrated when we discovered that the gene coding for RNF168 is mutated in the RIDDLE syndrome, an immunodeficiency and radiosensitivity disorder.
The discovery of this pathway has raised a number of questions that we are actively pursuing. For example: how is this pathway activated? What are the substrates of these enzymes? How is this pathway integrated with other chromatin-modifying activities? Is this pathway involved in tumor suppression?
If we are ever to understand fully how cells deal with DNA damage, we need to discover all the proteins involved in the DNA damage response. While work in model organisms has been immensely successful in uncovering the core machinery of the DNA damage response, human genetics and unbiased functional genomic screens have clearly demonstrated the need for unbiased, phenotype-based gene discovery in the DNA damage response.
We are fortunate to be located in Toronto, where world-class colleagues and facilities exist. We have taken advantage of these resources to develop image-based, genome-scale RNA interference screens that probe various aspects of the DNA damage response. As a case and point, the discovery of both RNF8 and RNF168 were the consequence of these efforts (Kolas et al. 2007 Science; Stewart et al. Cell 2009). We have a number of additional screens in the pipeline along with additional hits from screens. Our hope is to continue to uncover and characterize important new pathways that modulate genome integrity in human cells.
We are very interested in understanding how cells deal with their chromosome ends – the telomeres. Although telomeres are essentially DNA double-strand breaks, it is well known from the seminal work of Müller and McClintock that telomeres that they are dealt differently from DSBs. Conversely, DSBs must not be recognized as telomeres since the action of telomerase on accidental DNA breaks has dire consequences for genome integrity.
A few years ago discovered an evolutionarily-conserved protein complex, called the KEOPS complex, which plays an important role in a number of aspects of telomere function (Downey et al. 2006 Cell). We are keen to understand this complex at a biochemical level and to understand the mechanism by which it exerts its effect on telomeres. The function of the KEOPS complex is very mysterious and elucidating it will likely unravel fundamental new insight on the biology of nucleic acids.
More recently, we have carried out a genetic screen in budding yeast to uncover new genes that are necessary to convert a DSB into a telomere. We identified the Rrd1 protein as being critical for telomere action on DNA breaks, and on DNA breaks only (Zhang & Durocher, 2010 Genes & Development). Rrd1 is a regulator of PP2A phosphatases and by some cunning detective work, Wei, a graduate student in the lab was able to discovery that the ATR homolog in yeast, Mec1, plays a key role in inhibiting the recruitment of a key telomerase regulator, Cdc13, on DSBs. In fact, Wei found that Mec1 phosphorylates Cdc13 and that Rrd1 antagonizes this phosphorylation. We are now very keen to decipher how Cdc13 is recruited to DSBs and Wei’s screen has also identified multiple additional genes that deserve our attention. The questions of how cells DNA-end fate is a fundamental question in chromosome biology that will keep us busy for years to come.