Research
Building a complete network of SLX4 complexes
Synopsis
SLX4 is a protein scaffold that provides the 'landing pad' for a plethora of proteins that have key roles in genome stability and normal cell function. Importantly, many of these protein-protein interactions are regulated spatially (e.g. sites of DNA damage) or temporally (e.g. mitosis). We are using cutting-edge proteomics to develop a comprehensive network of SLX4 complexes in human cells. Our studies will define the SLX4 interactome in different phases of the cell cycle, as well as in cells that have been treated with clinically relevant DNA-damaging agents. These studies will increase our understanding of the identity, assembly, and functions of macromolecular SLX4 complexes in human cells.
Regulating the promiscuous SLX1 nuclease
Synopsis
Structure-selective endonucleases cleave DNA secondary structures that form during cell growth and DNA repair. The removal of these structures is essential for genome stability. Nevertheless, the act of cutting DNA is a double-edged sword because it can fuel genetic rearrangements commonly observed in cancer. SLX1 is unique among the structure-selective endonucleases because it exhibits almost no structure-selectivity in vitro. This raises an interesting question as to how cells prevent this promiscuous nuclease from catalyzing rampant cleavage of genomic DNA. We are taking a multi-pronged experimental approach to address this fascinating question. Stay tuned for more!
How to make and break the SMX tri-nuclease
Synopsis
The Wyatt lab studies three structure-selective endonucleases, called SLX1-SLX4, XPF-ERCC1 and MUS81-EME1, that have essential roles in DNA repair and genome stability. In human cells, the SLX4 subunit provides a scaffold for the assembly of these enzymes into at least two different macromolecular complexes: 1) SLX1-SLX4-XPF-ERCC1, which we refer to as the SX complex and 2) SLX1-SLX4-MUS81-EME1-XPF-ERCC1, also known as the SMX complex. Of note, MUS81-EME1 is recruited to the SX complex in early mitosis. We also know that the temporally regulated assembly of SMX is important for chromosome stability. We are currently using biochemical and microscopy- based experiments to determine how the SMX complex assembles and disassembles in human cells.
Mechanism of MUS81-EME1 activation in SMX
Synopsis
In the Wyatt lab, our main goal is to understand the structure, function, and interactions of proteins and nucleic acids that enable life. One particularly interesting enzyme is a multi-protein complex called SMX, built from three structure-selective endonucleases called SLX1-SLX4, MUS81-EME1, and
XPF-ERCC1. Interestingly, MUS81-EME1 is 'activated' upon recruitment to SMX, such that it becomes the predominant nuclease within this complex. We are currently using biochemical and biophysical approaches to determine how MUS81-EME1 is activated within SMX. These studies will reveal the mechanisms that underpin the enzymatic activities of the SMX tri-nuclease.
Characterization of novel SLX4-nuclease complexes
Synopsis
We hypothesize that SX and SMX have non-overlapping activities that reflect specialized roles at distinct phases of the cell cycle. This question cannot be addressed with cell-based experiments alone because of the functional redundancy observed between different structure-selective endonucleases. Instead, the Wyatt lab is perfectly situated to test this hypothesis using biochemical assays with purified enzymes and DNA substrates that represent different types of branched DNA structures. Since an understanding of protein structure often reveals critical information about protein function, the Wyatt lab also collaborates with top-notch structural biologists that specialize in NMR (Prof. Cheryl Arrowsmith and Prof. Simon Sharpe), crystallography (Prof. Trevor Moraes) and cryoEM (Prof. Marcin Nowotny). Of note, all of our purification strategies are designed in a forward-thinking manner so that we can readily integrate novel SLX4-binding partners into these workflows. Stay tuned to learn more about the structure and function of macromolecular nuclease complexes!
How is SLX4 (and friends) recruited to chromatin?
Synopsis
Protein-protein interactions have pivotal roles in the cellular functions and biological processes of all organisms. Although the requirement for SLX4-nuclease complexes in DNA repair and genome stability is well-established, we know surprisingly little about how these complexes are recruited to chromatin. Through an ongoing collaboration with Prof. Anne-Claude Gingras, we have identified several interesting candidates that could regulate the recruitment of SLX4 (and friends) to chromatin in human cells. Our next goal is to test this hypothesis using complementary biochemical and cell-based approaches. We can also use ChIP-seq to determine if there are regions in the human genome that represent nuclease 'hot-spots'. Stay tuned to learn more about the cellular substrates of structure-selective endonucleases, and whether these binding sites correlate with regions that are prone to genetic instability in cancer cells.