High-resolution genomic analysis of helicase loading to uncover how replication origins are recognised, regulated and function for genome stability

Our overall aim is to discover how replication origins function and are regulated. To gain insight into the protein-DNA interactions that occur at replication origins, we employ cutting-edge genomic approaches. In order to maximise the resolution of our genomic data, we employ novel methods such as ChIP-exo and optimised crosslinking-resistant affinity tags. Thus, we can generate base-pair resolution data that have been obscure to traditional ChIP-seq approaches. We combine the ChIP-exo data sets with proteomic data and AlphaFold-multimer predictions to understand the multi-protein complexes assembled at these sites and define how replication origins function. In consequence, we can predict how cancer-associated mutations affect replication origin function. This combination of methods we have termed “structural genomics”, an approach that sets us apart from any other genomic research groups.

We have recently used this approach to uncover how budding yeast replications function (for more details, see below) and started to use structural genomics in the context of human DNA replication.

MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing

Origin recognition complex (ORC)-dependent loading of the replicative helicase MCM2-7 onto replication origins in G1-phase forms the basis of replication fork establishment in S-phase. However, how ORC and MCM2-7 facilitate genome-wide DNA licensing is not fully understood. Mapping the molecular footprints of budding yeast ORC and MCM2-7 genome-wide, we discovered that MCM2-7 loading is associated with ORC release from origins and redistribution to non-origin sites. Our bioinformatic analysis revealed that origins are compact units, where a single MCM2-7 double hexamer blocks repetitive loading through steric ORC binding site occlusion. Analyses of A-elements and an improved B2-element consensus motif uncovered that DNA shape, flexibility, and the correct spacing of two face-to-face binding-elements as hallmarks of ORC-binding and efficient helicase loading sites. Thus, our work identified fundamental principles for MCM2-7 helicase loading that explain how origin licensing is realised across the genome.

ChIP-exo analysis of yeast MCM2-7 binding
Figure 1. ChIP-exo analysis of yeast MCM2-7 binding

Reuter, L. M., Khadayate, S. P., Mossler, M., Liebl, K., Karimi, M. M., Speck, C. (2023). MCM2-7 loading-dependent ORC release ensures genome-wide origin licensing. Nature Structure & Molecular Biology, (in revision).

If you are interested in using this approach, please contact Christian Speck.  We have an open postdoctoral position to investigate human DNA replication using “structural genomics”.