<i>Supporting data for “Mechanistic Study of the Role of Orc2 in Replication Licensing”</i>

<i>This is the supporting data for the thesis: “Mechanistic Study of the Role of Orc2 in Replication Licensing”. </i><i>Data is categorized according to the experimental assays.</i><i>This thesis focused on studying the molecular process of replication initiation. At replication origins, ORC activity is strictly regulated to ensure </i><i>the assembly of pre-replication complex (pre-RC) at the right place and </i><i>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 </i><i>has shown that Orc2 has a highly flexible N-terminal domain (NTD), </i><i>which contains six S-CDK phosphorylation sites and a few DNA-binding </i><i>basic patches (BPs). However, the role of Orc2-NTD in replication licensing remains elusive. This study aims to elucidate how Orc2 regulates </i><i>MCM loading at the origin DNA via its flexible NTD.</i><i>In this study, we identified a motif in the yeast Orc2-NTD that is </i><i>essential for cell growth. Using in vivo approaches, we found that Orc2-</i><i>NTD is required to promote an efficient initiation of DNA replication (data in folder ../cell_analysis). </i><i>Further analyses utilizing in vitro and in vivo Mcm2-7 loading assays </i><i>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 </i><i>the genome. In addition, negative staining electron microscopy (NS-EM) imaging showed an accumulation of the OCCM-like intermediate </i><i>in pre-RC assembly reactions with Orc2-NTD mutants (data in folder ./NS2D). This defect was </i><i>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-</i><i>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 </i><i>pull-down assays and photo-crosslinking mass spectrometry (XL-MS) to </i><i>identify its interacting partners (data in folder ./pulldown_analysis and ./massspec, respectively). We found that Orc2-NTD specifically </i><i>binds to Mcm2 on a surface proximal to the Mcm2/Mcm6 ATPase interface. In addition, our results indicate that S-CDK targets Orc2-NTD </i><i>for inhibitory phosphorylation to interrupt the interaction between Orc2 </i><i>and Mcm2.</i><i>Taken together, our study unravels a novel mechanism by which </i><i>ORC promotes MCM loading by utilizing the highly flexible region of </i><i>Orc2-NTD to enhance the ATPase activity of Mcm2-7. Phosphorylation </i><i>of Orc2 by S-CDK inhibits this process, to prevent excessive licensing of </i><i>origins.</i><br><br>