Publications
Learn more about our research by checking out our publications below. We hope you find them as interesting as we do!
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Primary Source Articles
Phosphorylation of the DNA repair scaffold SLX4 drives folding of the SAP domain and activation of the
MUS81-EME1 endonuclease
Summary: The DNA repair scaffold SLX4 has multifaceted roles in genome stability, many of which depend on structure-selective endonucleases. SLX4 coordinates the cell cycle-regulated assembly of SLX1, MUS81-EME1, and
XPF-ERCC1 into a tri-nuclease complex called SMX. Mechanistically, how the mitotic kinase CDK1 regulates
the interaction between SLX4 and MUS81-EME1 remains unclear. Here, we show that CDK1-cyclin B phosphorylates SLX4 residues T1544, T1561, and T1571 in the MUS81-binding region (SLX4 MBR). Phosphorylated SLX4 MBR relaxes the substrate specificity of MUS81-EME1 and stimulates cleavage of replication and recombination structures, providing a biochemical explanation for the chromosome pulverization that occurs when SLX4 binds MUS81 in S-phase. Remarkably, phosphorylation of the SLX4 MBR drives folding of an SAP domain, which underpins the high-affinity interaction with MUS81. Our work provides mechanistic insights into how cell cycle-regulated phosphorylation of SLX4 drives the recruitment and activation of MUS81-EME1.
An open-source platform to quantify subnuclear foci and protein colocalization in response to replication stress
Summary: Nuclear reorganization, including the localization of proteins into discrete subnuclear foci, is a hallmark of the cellular response to DNA damage and replication stress. These foci are thought to represent transient environments or repair factories, in which the lesion is sequestered with molecules and co-factors that catalyze repair. Here we describe an open-source and semi-automated method to detect and quantify subnuclear foci, as well as foci colocalization and the accompanying pixel-based colocalization metrics. We use this pipeline to analyze the composition of subnuclear foci that form in pre-mitotic nuclei. Altogether, our results point towards a role for SLX1-SLX4 and XPF-ERCC1, but not MUS81-EME1, in the early cellular response to replication stress.
Interested in using these methods? We posted our programming scripts and step-by-step instructions in Wyatt Lab Spells.
The SMX DNA repair tri-nuclease
Summary: Backed by the momentum of our previous work (Wyatt et al 2013), here we explore the assembly and biochemical properties of a tri-nuclease complex called SMX. This study provides the first insight into the enzymology of a tri-nuclease complex. We show that the SLX4 scaffold activates the MUS81-EME1 nuclease to cleave fork-like DNA structures, revealing a novel mechanism of MUS81-EME1 regulation.
Co-ordinated actions of SLX1-SLX4 and MUS81-EME1 for Holliday junction resolution in human cells
Summary: This work reveals a cell cycle-dependent nuclease complex minimally comprised of SLX1-SLX4 and MUS81-EME1. We elucidate the mechanism by which the SLX4 scaffold co-ordinates the SLX1 and MUS81-EME1 nuclease active sites during Holliday junction resolution. Our work also highlights the biological ramifications of unresolved Holliday junctions.
Structural and mechanistic analysis of the Slx1-Slx4 endonuclease
Summary: In a fruitful collaboration led by Marin Nowotny, we provide the first structural details for Slx1, alone and bound to Slx4. We show that Slx1 forms a catalytically-inactive homodimer in the absence of Slx4. Our data also reveal a RING domain in the Slx1 C-terminus. These observations reveal intriguing mechanisms that may regulate Slx1-Slx4 activity.
Literature Reviews
Exploring the structures and functions of macromolecular SLX4-nuclease complexes in genome stability
Summary: This review focuses SLX1-SLX4 and the
nucleases that bind the SLX4 scaffold. First, we discuss the structure and biochemical properties of this atypical structure-selective endonuclease. We then summarize the multifaceted roles that are fulfilled by human SLX1-SLX4 and its associated endonucleases in homologous
recombination and genome stability. Finally, we discuss recent work on SLX4-binding proteins that may represent integral components of these macromolecular nuclease
complexes, emphasizing the structure and function of a protein called SLX4IP.