| Popis: |
Prior to each cell division, genomic information must be duplicated precisely once to support cell proliferation and to preserve normal cell function. Hence, over- or under-replication of the genome can have detrimental consequences such as carcinogenesis and developmental disorders. Across the tree of life, DNA replication initiates from discrete sites (also known as origins) to achieve timely genome duplication. Origins are recognized by dedicated initiator proteins that collaborate with co-factor proteins to load replicative helicases onto DNA. Replicative helicases are essential for unwinding the DNA and constitute the core of the DNA replication machinery. In bacteria, archaea and a few eukaryotes, like S. cerevisiae, origins are defined by specific DNA sequence motifs that direct initiator binding. In contrast, metazoan origins are not characterized by a DNA consensus sequence but by contextual factors in cis (like DNA structural properties and chromatin environment) and trans (protein factors), and the metazoan initiator Origin Recognition Complex (ORC) is known to bind DNA independent of a consensus motif. However, it is a long-standing question how metazoan ORC recognizes origin DNA. In my Ph.D., I used single-particle cryo-EM to determine high-resolution structures of Drosophila ORC bound to different DNAs in presence or absence of the co-loader Cdc6. These structural studies provided insight into how the metazoan initiator and co-loader recognize DNA and help explain how DNA binding by ORC regulates initiator activity. Upon binding by the initiator, origins are remodeled to support efficient helicase loading. Origin remodeling by the initiator is likely influenced by intrinsic DNA sequence properties and local chromatin structure, but it is unclear how these factors contribute to initiator recruitment and helicase loading at origins in metazoans. We established a metazoan helicase loading assay in vitro and combined it with biochemical assays and cryo-EM to investigate how DNA sequence affects initiator binding, DNA remodeling and replicative helicase loading. We found that DNA sequence properties, such as minor groove width and negative electrostatic potential, contribute to metazoan ORC binding affinity and that metazoan ORC binding to a DNA substrate can be uncoupled from ORC’s ability to bend DNA. However, we show that DNA bending by ORC is crucial for efficient helicase loading. Hence, we suggest that DNA shape is an important factor in origin selection and licensing in metazoans. Initiation of DNA replication is integrated into the cell cycle to ensure that all genomic information is duplicated exactly once before cells divide. Cell-cycle dependent kinases (CDK) phosphorylate initiation and loading factors to regulate their activities, thereby coordinating the initiation of DNA replication with diverse cellular processes. Although phosphorylation-dependent regulation of initiation factors is well established, the underlying structural mechanisms are not well defined. Furthermore, origin licensing requires that the initiator, co-loader and helicase proteins undergo large conformational changes that are tightly coupled to ATP binding and hydrolysis. We have just begun to understand how these dynamic structural rearrangements are regulated, and the physical basis for coordinated nucleotide binding and hydrolysis is largely unclear. We determined high-resolution cryo-EM structures of the ternary budding yeast ORC-DNA-Cdc6 complex to understand better the physical basis by which CDK-dependent phosphorylation regulates initiator activity and to investigate how the ternary ORC-DNA-Cdc6 assembly coordinates ATP hydrolysis. We propose a structural mechanism for coordinated ORC/Cdc6 ATPase activity and report a potentially autoinhibited conformation of the budding yeast ORC-DNA-Cdc6 complex that appears to be incompatible with helicase loading. Biochemical analysis suggest that ORC is phosphorylated in our ORC-DNA-Cdc6 structure, suggesting that we have uncovered a structural explanation of how CDK-dependent phosphorylation of ORC inhibits helicase loading. Metazoan initiation factors are delicate multi-protein assemblies that have evolved highly-functionalized domains to support timely DNA replication. Amino acid mutations in these specialized initiator domains have been shown to severely impair human development leading to the Meier-Gorlin Syndrome (MGS), but our molecular understanding of how these mutations derail initiation factor function is limited. We have purified metazoan ORC harboring MGS-mutations and characterized their functional consequences using biochemical tools in vitro. We find that MGS-associated mutations in Drosophila ORC compromise different initiator functions to varying degrees, which helps define the molecular basis for MGS. Taken together, the studies presented here advance our knowledge of how metazoan origins are defined on a molecular level and how these characteristics might be read out by the initiator to facilitate origin selection and efficient helicase loading. Furthermore, our comprehensive structural characterization of Drosophila and budding yeast ORC in complex with DNA and Cdc6 reveals dynamic conformational rearrangements that are necessary for helicase loading and suggest mechanisms for the controlled hydrolysis of ATP by the initiator and its co-loader. Moreover, we unveiled an unknown conformational state of the budding yeast ORC-DNA-Cdc6 complex that provides a structural explanation for how phosphorylation of ORC regulates origin licensing. Finally, our functional characterization of MGS mutations in ORC aids our understanding of the molecular mechanism underlying this developmental disease. The in vitro metazoan helicase loading assay established by my work will facilitate future research efforts to explore how origin licensing is regulated in higher eukaryotes. |
