Haley Wyatt

Haley Wyatt

Assistant Professor

BSc Hons, University of Regina, 2004
PhD, University of Calgary, 2009
Postdoc, The Francis Crick Institute, 2017

Address MaRS Center, West Tower, Room 1521A
661 University Avenue
Toronto, ON M5G 1M1
Lab Wyatt Lab
Office Phone 416-978-4808
Email haley.wyatt@utoronto.ca

Haley Wyatt was born and raised in the small rural community of Broadview, Saskatchewan (Canada). Her research training in biochemistry and molecular biology began during her postgraduate studies at the Southern Alberta Cancer Research Institute and the University of Calgary. Under the supervision of Tara Beattie, she studied the human telomerase reverse transcriptase (hTERT). hTERT is the catalytic subunit of telomerase, an enzyme that has a pivotal role in cellular proliferation and organismal aging and is deregulated in many types of cancer. Haley developed the first biochemical assay to study interactions between hTERT and telomeric DNA substrates. This assay was critical for subsequent structure-function studies, in which she provided important insight into the molecular mechanisms of telomerase deficiency associated with human disease.

After receiving her PhD in 2009, Haley moved to Stephen West’s laboratory at the Francis Crick Institute (formerly Clare Hall Laboratories) in London, England to pursue her interests in DNA repair and mechanisms of genome instability. Her research has significantly advanced our understanding of the biochemical and cellular functions of the SLX4 protein and its role as a scaffold for an endonuclease DNA repair complex. This research is of particular relevance to human health because mutations in SLX4 (and its associated nucleases) are linked to Fanconi anemia, a complex disorder characterized by bone marrow failure, chromosomal instability, and cancer susceptibility. In 2017, Haley will move back to Canada to open her lab in the Department of Biochemistry.

Research Lab

The Wyatt lab officially opened on April 1, 2017! We will study the structure, function, and regulation of nucleases. These enzymes are scissors that have critical roles in repairing damaged DNA and maintaining genome stability. The lab will use a powerful combination of techniques in biochemistry and molecular biology, including protein expression and purification, enzymology, proteomics, microscopy, and phenotypic analyses of cultured human cells.

Research opportunities are available for enthusiastic, positive, and highly-motivated individuals that seek to understand how human cells maintain genome stability, and how protein dysfunction gives rise to human disease. Successful candidates will have an exciting opportunity to help build the Wyatt lab from the “ground-up” and will benefit from working alongside the Principal Investigator, who will provide them with direct mentorship and training.

Research Description

DNA Scissors, DNA Repair and Genome Stability

DNA Repair and Genome Stability

An essential component of every living organism is DNA, which provides the blueprint for life and is required for the biological processes in each and every cell. During normal cell growth, this genetic information must be accurately replicated and propagated to two daughter cells. Paradoxically, DNA is highly susceptible to damage by agents that occur naturally (e.g. metabolic by-products) and in the environment (e.g. ultraviolet radiation, carcinogenic chemicals). If left unrepaired, damaged DNA can trigger mutations, chromosomal rearrangements and genome instability. To counteract the deleterious effects of genotoxic agents, cells contain sophisticated DNA repair networks that safeguard genome integrity and ensure proper cell function. Understanding the intricate mechanisms that underpin these essential cellular pathways is a major goal of scientists that study the basic biological processes of DNA repair and genome stability.

DNA Scissors

Most DNA repair pathways require the actions of structure-selective endonucleases (SSEs), which are molecular scissors that remove potentially toxic DNA structures that form during DNA repair (and normal cell growth). The failure to remove these structures compromises chromosome stability. Nevertheless, DNA cleavage opens the door for indiscriminate repair that can fuel genetic rearrangements, emphasizing the importance of regulatory mechanisms to control the activity of SSEs and prevent uncontrolled DNA cleavage.

The conserved SSEs SLX1-SLX4, MUS81-EME1 and XPF-ERCC1 are required for DNA recombination and repair in most eukaryotes. The SLX4 protein provides a scaffold for the SMX tri-nuclease complex, formed by interactions with SLX1, MUS81-EME1 and XPF-ERCC1. SLX4 interacts with several other genome stability proteins, leading to the prevailing model that SLX4 provides a hub for the assembly of versatile macromolecular complexes that orchestrate diverse protein-DNA transactions. Key questions about the structure, function, and regulation of these complexes need to be addressed for a complete understanding of how these enzymes mediate genome stability.

Research Goals

The overarching aim of my lab is to elucidate the cellular roles, regulation and biochemical mechanisms of macromolecular SLX4 complexes. This information will provide a mechanistic framework for understanding how these complexes function and preserve genome integrity. This objective is inherently multi-disciplinary in nature and will ultimately involve structural, biochemical and cellular studies, thus providing an ideal training environment for researchers at all stages of their careers.

Define the Biochemical Mechanisms of SLX4-Nuclease Complexes in DNA Interstrand Crosslink (ICL) Repair

Research Context

Mutations in SLX4/FANCP and XPF/FANCQ are associated with Fanconi anemia (FA), a rare genetic disease characterised by genome instability and cancer predisposition. At the molecular level, cells exhibit a high frequency of chromosomal aberrations and an exquisite hypersensitivity to agents that cause DNA interstrand crosslinks (ICLs). Crucially, ICLs covalently link two nucleotides on complementary strands of DNA, thereby imposing a physical block to DNA transcription and replication. Despite intense research, the fundamental mechanisms of ICL repair are still not completely defined. Lesion heterogeneity, pathway cross-talk and/or functional redundancy have made it extremely challenging to decipher the specific biological role(s) of proteins implicated in ICL metabolism. Well-defined biochemical systems are needed to elucidate the precise mechanisms of ICL repair.

Hypothesis & Significance

The hypothesis of this aim is that SLX4 orchestrates the dynamic assembly of nucleases to catalyse ICL repair; the nuclease(s) involved depend on the degree of helical distortion imposed by the ICL, as well as the DNA structure within which the ICL is embedded. This hypothesis will be addressed with systematic, in vitro analyses of biologically relevant SLX4-nuclease complexes and crosslinked DNA structures. Determining the mechanism(s) by which these complex lesions are repaired will provide unprecedented mechanistic insights into ICL repair.

Elucidate Mechanisms of SLX1 Regulation in DNA Repair and Genome Stability

Research Context

Despite tremendous advancements in our understanding of the cellular functions of human SLX4-nuclease complexes in DNA repair, we have much less information on the precise biochemical mechanisms by which these complexes execute their functions. Another key area of research that remains to be fully elucidated concerns the mechanisms that regulate temporal and spatial aspects of macromolecular SLX4-nuclease complexes. For example, virtually nothing is known regarding SLX1 regulation. This is surprising given the promiscuous nuclease activity of SLX1-SLX4 and its potential to trigger widespread DNA cleavage and genetic instability.

Hypothesis & Significance

We hypothesize that SLX1 is regulated by protein-protein interactions and/or post-translational modifications. This will be addressed through three specific aims, as detailed below:

  1. Regulation of SLX1 nuclease activity by inhibitory homodimerization. It is well established that SLX1 nuclease activity depends on its interaction with SLX4. Our recent structural studies of Candida glabrata Slx1-Slx4 revealed critical insights into the underlying mechanism (Gaur et al., 2015). We showed Slx1 is a stable homodimer in the absence of Slx4, an architecture that obscures key catalytic and DNA-binding residues. The presence of Slx4 triggered the displacement of the homodimer, leading to the formation of a catalytically active Slx1-Slx4 heterodimer. This led us to propose a novel mechanism of Slx1 regulation, namely inhibitory homodimerization. The goals of this project are two-fold: i) determine if human SLX1 is regulated by inhibitory homodimerization and ii) generate an SLX4-independent nuclease to alleviate the cellular defects exhibited by human cells lacking the SLX4 scaffold.
  2. SLX1 regulation by cell cycle-stage specific partner proteins. The human SLX1 ‘interactome’ will be characterized using cutting-edge complementary proteomics methods: i) biotin ligase-based proximity tagging (BioID) and ii) affinity purification (AP)-mass spectrometry (MS) combined with stable isotope labelling with amino acids in cell culture (SILAC). These studies are expected to identify novel SLX1-binding proteins and provide important new insights into SLX1 function and regulation.
  3. Role for the SLX1 RING domain in ubiquitylation. One interesting feature of SLX1 is the presence of a C4HC3-type RING domain, typically found in E3 ubiquitin ligases (Gaur et al., 2015). Ubiquitin E3 ligases catalyse the final reaction in the E1-E2-E3 ubiquitylation cascade, promoting the transfer of ubiquitin from an E2 conjugating enzyme to a lysine residue in the target substrate. We hypothesize that the SLX1 RING domain confers SLX1-SLX4 with an E3 ligase activity that targets SLX4-bound substrates for ubiquitylation, which regulates key processes that maintain genome stability. For example, SLX1 autoubiquitylation could negatively regulate its promiscuous nuclease activity. This project will expand the functional repertoire of the SLX1-SLX4 complex, and elucidate a novel mechanism that regulates DNA repair and genome stability.

Identify & Characterise Novel SLX4 Complexes in Response to Genotoxic Agents

Research Context

Human SLX4 provides a docking platform for diverse proteins, through which SLX4 mediates essential biological processes. Despite recent advancements in our understanding of the cellular functions of SLX4 in DNA repair, the mechanistic details that underpin these functions are still incomplete. Another key area of research that remains to be fully elucidated concerns the mechanisms that regulate temporal and spatial aspects of macromolecular SLX4 complexes. This is partly due to challenges associated with studying the dynamic and transient molecular events that occur during DNA repair. In addition, it is clear that the proteins and enzymes involved in DNA repair are tailored to specific cellular needs through flexible molecular switches that regulate protein function and/or interactions with other partner proteins. Innovative proteomics techniques have been developed to help researcher’s address these difficult-to-study questions.

Hypothesis & Significance

The overarching hypothesis is that distinct macromolecular SLX4 complexes form in response to different cellular stimuli. This project will generate the first comprehensive and quantitative protein-protein interaction maps for the human SLX4 scaffold, in response to clinically relevant DNA-damaging agents that activate distinct DNA repair pathways (i.e. cisplatin, camptothecin, olaparib). This objective will be achieved using two complementary mass spectrometry-based techniques: i) biotin ligase-based proximity tagging (BioID) and ii) affinity purification (AP)-mass spectrometry (MS) combined with stable isotope labelling with amino acids in cell culture (SILAC). This project is critical to advance our understanding of the mechanisms that regulate the cellular response to such compounds, as well as the mechanisms that regulate the SLX4 scaffold.


Awards & Distinctions

2017 - 2022 — NSERC Discovery Grant


View all publications on PubMed