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Projects

Huilin Li Laboratory

Eukaryotic DNA replication

Eukaryotic chromosomal replication initiation is an intricate process that requires the coordinated and tightly regulated action of numerous molecular machines. Failure to ensure once-only replication initiation per cell cycle can result in uncontrolled proliferation and genomic instability, two hallmarks of tumor genesis. projects - figure 1The origin recognition complex (ORC), first discovered in yeast by our collaborator Dr. Bruce Stillman’s lab, is a six-protein machine conserved in all eukaryotes. Yeast ORC constitutively binds to and marks the replication origin throughout the cell cycle. Licensing of the DNA replication origin starts when the critical cell division cycle protein Cdc6p binds to ORC in the G1 phase of the cell cycle. Work in my lab has elucidated several key steps in origin activation and Mcm2-7 hexamer recruitment on to DNA. We have revealed the architecture of ORC, how Cdc6 completes the ORC ring and activates it for subsequently loading of the replicative helicase, how ORC-Cdc6 binds and cracks open and then loads the Cdt1-bound Mcm2-7 helicase core onto the origin DNA, how the two Mcm2-7 hexamers come together to form the pre-replication complex—the Mcm2-7 double-hexamer. In the S phase of the cell cycle, the active CMG helicase (CMG) works with the leading strand polymerase epsilon, the lagging strand polymerase delta, and the primase-polymerase alpha to synthesize new DNA. In collaboration with Michael O’Donnell, we have determined the structure of the 11-protein yeast CMG helicase (Figure: Cryo-EM structure of the CMG helicase in a side view), and mapped a few key components of the replisome. Our work has advanced the field of eukaryotic DNA replication.

Mycobacterium tuberculosis Pup-proteasome system

projects - figure 2Tuberculosis kills 1.5 to 2 million people globally every year. An effective vaccine or chemotherapy has yet to be developed. Recently, through a large-scale transposon mutagenesis screening, the Mycobacterium tuberculosis (Mtb) proteasome and Mtb proteasomal ATPase (Mpa) were found to be required for Mtb resistance to killing by a source of nitric oxide (NO). NO is required by the host immune system to control Mtb infections. Proteasome and Mpa appear to protect Mtb against NO by degrading proteins after exposure to NO. Thus, Mpa and the Mtb proteasome may be promising targets for the development of anti-Tb chemotherapeutics. We have combined cryo-EM, X-ray crystallography, and protein biochemistry to elucidate the structure and function of the bacterial proteasome system. We found that the Mtb proteasome is structurally similar to the eukaryotic counterparts yet possess unique assembly and gating mechanism. We elucidated the structural basis for species-specific inhibition of the Mtb proteasome inhibitor Oxathiazol-2-ones. We further revealed that the protein degradation tag Pup, a prokaryotic ubiquitin-like protein, is intrinsically disordered, but folds into an α-helix upon binding to and recognized by the proteasomal ATPase Mpa (Figure: yellow – 20S proteasome, green and purpule: Mpa atomic model, red, Pup). Our work is setting the stage for the structure-based anti-TB chemotherapeutic development.

Regulation and modification of the Notch receptor

projects - figure 3Notch is a major receptor that controls the development of multicellular organisms. It plays an important role in how cells sense their neighbors and, by controlling gene expression, determines which cells should develop into different types and how much they should grow-like a master controller. We have recently focused on enzymes the add a glucose-xylose-xylose trisaccharide on Notch. We have solved the structure of Rumi that adds the first glucose to Notch and found that Rumi recognizes a six-amino-acid sequence on many of the 36 EGF motifs of Notch and a hydrophobic patch. Our study suggested that mutated forms of Rumi could be the cause of cancers because these mutant enzymes are unable to modify Notch (Figure: a, Rumi in complex with acceptor EGF and donor UDP-Glc. Cancer related mutations are shown in spheres. B. Mutations found in cancers and Dowling-Degos disease abolish Rumi activity in vitro). This observation links Rumi defects with cancers. XXYLT1 is a retaining glycosyltransferase that adds the third surgar of the trisaccharide—a xylose—to Notch. The chemical mechanism for retaining enzymes such as XXYLT1 had been controversial in the field for several decades. Our structural studies of the XXYLT1 unambiguously supported the SNi-like retaining mechanism and disproved the double displacement mechanism. Our studies provide essential information for the design of inhibitors that could potentially become therapies for Notch-related diseases.