New publication in Nature Communications

Our latest work using peptide tools to understand DNA replication is out in Nature Communications

This project was the work of former PhD student in the group, Joshua Tomkins, working in collaboration with Christian Speck and Alexis Barr at the Laboratory of Medical Sciences (LMS). We used peptides mimicking sections of the protein geminin to understand how it regulates DNA replication - an important cancer target. 

DNA replication is tightly regulated to occur once per cell cycle, with the MCM2-7 helicase loaded onto replication origins only during G1-phase. In higher eukaryotes, geminin negatively regulates this process during S-, G2- and M-phases by binding the essential licensing factor CDT1. Although geminin’s function is crucial for genomic stability, its inhibitory mechanism remains elusive. Here, we utilise a fully reconstituted human DNA replication licensing assay to dissect geminin’s role. AlphaFold modelling provides structural insights into an N-terminal CDT1-binding helix of geminin, which proves essential for inhibition. Structural docking of the CDT1-geminin complex into the ORC-CDC6-CDT1-MCM2-7 (OCCM) assembly shows that geminin’s long coiled-coil domain sterically clashes with the MCM2 C-terminus, rather than directly blocking CDT1 binding to ORC-CDC6-MCM2-7. Shortening the coiled-coil preserves geminin dimerisation and CDT1 binding but abolishes inhibition, confirming its mechanistic role. Surprisingly, geminin is not able to fully inhibit DNA licensing. However, CDK1/2-cyclin A can partially inhibit DNA licensing and, in conjunction with geminin, result in a complete block. These findings uncover geminin’s steric inhibitory mechanism and suggest that a dual CDK-geminin axis controls human DNA replication.