<b>Supporting Data for Molecular Mechanism of Replicative Helicase Activation in Human Cells</b>

<p dir="ltr">In eukaryotes, DNA replication is strictly regulated to achieve a complete and accurate genome duplication. The initiation of DNA replication requires two distinct and temporally separated stages: replication licensing and origin firing. During the first stage, two copies of minichromosomal maintenance 2-7 (MCM 2-7) are loaded onto replication origins to form MCM 2-7 double hexamers (MCM-DHs), a process known as pre-replication complex (pre-RC) assembly. In G1 phase of the cell cycle, MCM-DHs remain inactive in their helicase activity. Upon entering S phase, pre-RCs are further activated with the help of kinases DBF4-dependent kinase CDC7 (DDK) or Cyclin B-CDK1 together with other origin firing factors, forming the pre-initiation complex (pre-IC) at origin DNA upon which two sister replisomes are established.</p><p><br></p><p dir="ltr">In the past decades, numerous efforts have been devoted to investigating how DNA replication is initiated in the model organism budding yeast. However, in human cells, the mechanisms by which the activities of various replication factors are synergized at origin DNA to reconfigure the MCM-DHs for helicase activation and subsequent replisome establishment remain largely unknown.</p><p><br></p><p dir="ltr">In this study, we aim to elucidate the molecular mechanism governing MCM-DH activation in human cells. First, we investigated the roles of the kinases acting on the human MCM-DH. To this end, we purified DDK and CCNB1-CDK1 and assembled the DH-DDK and DH-CCNB1-CDK1 complexes for biochemical analysis and structure determination using cryo-electron microscopy (cryo-EM). Our results demonstrate direct interactions between the purified kinases, DDK and Cyclin B-CDK1, and their substrate, MCM-DH, and robust phosphorylation events resulting from in vitro kinase assays. Notably, Cyclin B-CDK1 emerges as a unique kinase targeting MCM-DH, a mechanism not conserved in budding yeast. Our preliminary structural analysis shows obvious extra densities, corresponding to the kinase complexes, attached to the MCM-DH at its hexamer interface region, which appears highly dynamic. These results lay a solid foundation to further characterize the activities of both DDK and Cyclin B-CDK1 in helicase activation, providing key insights into our understanding of replication initiation in human cells.</p><p><br></p><p dir="ltr">To understand the interplay between kinase-phosphorylated MCM-DH and other firing factors, including CDC45, GINS, DONSON, MTBP, TRESLIN, and TOPBP1, during helicase activation, we sought to establish an in vitro helicase activation assay with purified human proteins for functional and structural studies. To date, using diverse protein expression systems, we have purified the relevant firing factors for assembly experiments. To identify novel factors involved in helicase activation, we also overexpressed all known firing factors in HEK293T cells, which the lysate will be incubated with the phosphorylated MCM-DHs. This approach will enable us to identify any potential factors assembled onto the MCM-DHs by kinases via mass spectrometry for further study using in vitro helicase activation assay.</p><p><br></p><p dir="ltr">The outcomes of this research are expected to provide valuable insights into the regulation of DNA replication initiation in human cells. The detailed structural frameworks derived from this study will be a wealth of information regarding specific protein-protein interactions or conformational changes that are critical for helicase activation. These results hold the potential for the design of inhibitors that can target those interactions specific for replication initiation to selectively kill cancer cells.</p>