Capturing Kinetically Labile Multiprotein Assemblies on DNA by Chemical Crosslinking

Type of Award: Catalyst
Date Awarded: January 2012
Award End Date: December 2013
Amount Awarded: $ 200,000.00
PI(s): Jung-Hyun Min, PhD, UIC; Chuan He, PhD, UChicago;

Abstract: Control and maintenance of the human genome is carried out by proteins that assemble at specific DNA sites, driven by highly specific and often labile interactions. These biologically critical interactions are difficult to study because of challenges in preparing large and complex protein machines and in assembling them on DNA in a stable manner for detailed characterization. We propose a strategy against these challenges by combining the unique strengths of two groups: The Min group (UIC) can produce complex proteins for biochemical and structural analysis using a new "MultiBac" system; and the He group (U of Chicago) uses chemical crosslinking technologies to stabilize protein-DNA and protein-protein interactions. We will apply our joined forces to determine the structures of previously intractable protein-DNA complexes, focusing on detailed biochemical characterization of binding events, to answer questions such as: how do proteins assemble in the right place on the DNA out of myriad possible locations? What are the architectures of the assembly that control the biological process? We select two areas of the highest scientific interest to showcase our approach:

(1) All cells rely on efficient DNA damage repair complexes such as XPC, yet it is unknown how this recognizes DNA lesions within a vast genome. We will capture XPC on DNA and analyze the assembly events during the damage repair process.

(2) DNA hydroxymethylation by TET-family enzymes has recently been shown as critical epigenetic marking that controls gene regulation. Yet how TET locates and processes target DNA sites is unknown. We will trap and characterize TET and its cofactor proteins during this vital epigenetic operation. Our success will represent major discoveries in genetic mechanisms, create a long-term collaboration between a junior (Min) and senior faculty (He) in CBC and lead to joint federal grants for applying the approach to diverse biological systems.