Optimizing Treslin-MTBP for Cryo-EM Analysis

Author: Ng, Cai Tong

Year: 2026

Degree: Dissertation (Ph.D.)

Advisor: Dunphy, William G.

Committee Members: Rees, Douglas C.; Chan, David C.; Glover, David M.; Dunphy, William G.

Option: Biochemistry and Molecular Biophysics

Abstract

DNA replication initiation is a highly regulated process in eukaryotes to maintain genome integrity. Misfiring of the DNA replication machinery from replication origins can result in gene amplification and genome instability. In budding yeast, ORC, Cdc6, Cdt1, and Mcm2-7 assemble on the sites of origins to form the inactive pre-replicative complex. Next, phosphorylation of Sld2 and Sld3 causes them to interact with Dbp11, leading to the recruitment of Cdc45 and GINS to the pre-replicative complex, which results in its activation. The vertebrate homolog of Sld3 is Treslin. Treslin is tightly associated with MTBP throughout the cell cycle, and the complex is essential for DNA replication in metazoan systems, including Xenopus egg extracts and human cells. Here, I aim to understand the mechanism of Treslin-MTBP function by studying its molecular structure. I established purification protocols to produce the Treslin-MTBP complex at high concentration. I then studied the overall structure of the complex using cryo-electron microscopy and obtained reconstructions of the complex at ~20 Å. The reconstructions show that the Treslin-MTBP complex exhibits a large variety of conformations, indicating its intrinsic flexibility. The complex is composed of large, stable domains that can be observed in 2D classes. Using cross-link mass spectrometry, I also showed that Treslin-MTBP transiently binds to Mcm4 in cells, making Mcm4 a potential binding partner for future structural studies of the complex.