Supporting data for “Mechanistic Study of the Role of Orc2 in Replication Licensing”

This is the supporting data for the thesis: “Mechanistic Study of the Role of Orc2 in Replication Licensing”. Data is categorized according to the experimental assays.

This thesis focused on studying the molecular process of replication initiation. At replication origins, ORC activity is strictly regulated to ensure the assembly of pre-replication complex (pre-RC) at the right place and time. The activity of S-phase cyclin-dependent kinase (S-CDK) toward the ORC inhibits the licensing process to prevent Mcm2-7 loading outside of G1-phase. The structure of the yeast ORC bound to origin DNA has shown that Orc2 has a highly flexible N-terminal domain (NTD), which contains six S-CDK phosphorylation sites and a few DNA-binding basic patches (BPs). However, the role of Orc2-NTD in replication licensing remains elusive. This study aims to elucidate how Orc2 regulates MCM loading at the origin DNA via its flexible NTD.

In this study, we identified a motif in the yeast Orc2-NTD that is essential for cell growth. Using in vivo approaches, we found that Orc2-NTD is required to promote an efficient initiation of DNA replication (data in folder ../cell_analysis). Further analyses utilizing in vitro and in vivo Mcm2-7 loading assays revealed that mutations in Orc2-NTD led to decreased pre-RC assembly on chromatin (data in folder ./MCM_loading), resulting in a failure of replication initiation across the genome. In addition, negative staining electron microscopy (NS-EM) imaging showed an accumulation of the OCCM-like intermediate in pre-RC assembly reactions with Orc2-NTD mutants (data in folder ./NS2D). This defect was accompanied by decreased ATPase activity of Mcm2-7 which is essential for efficient origin licensing (data in folder ./ATPase_assay). These observations suggest that Orc2-NTD plays an important role in regulating MCM loading by modulating ATP-driven conformational changes. To understand the molecular mechanism of Orc2-NTD in driving MCM loading, we performed pull-down assays and photo-crosslinking mass spectrometry (XL-MS) to identify its interacting partners (data in folder ./pulldown_analysis and ./massspec, respectively). We found that Orc2-NTD specifically binds to Mcm2 on a surface proximal to the Mcm2/Mcm6 ATPase interface. In addition, our results indicate that S-CDK targets Orc2-NTD for inhibitory phosphorylation to interrupt the interaction between Orc2 and Mcm2.

Taken together, our study unravels a novel mechanism by which ORC promotes MCM loading by utilizing the highly flexible region of Orc2-NTD to enhance the ATPase activity of Mcm2-7. Phosphorylation of Orc2 by S-CDK inhibits this process, to prevent excessive licensing of origins.