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Molecular and structural mechanisms for metazoan replication origin specification

Periodic Reporting for period 2 - ORISPECIFICATION (Molecular and structural mechanisms for metazoan replication origin specification)

Reporting period: 2020-01-01 to 2021-06-30

Cellular life depends on the timely and accurate duplication of chromosomal DNA through semi-conservative replication to sustain genomic integrity and organismal viability. In all domains of life, DNA replication relies on dedicated initiator proteins that recognize and bind specific genomic sites, termed replication origins, to facilitate the loading of ring-shaped replicative helicases onto DNA. While origin recognition by initiators is determined by specific DNA sequences in prokaryotes and in the eukaryote S. cerevisiae, origin specification in higher eukaryotes instead appears to rely on chromatin context and DNA structure. Yet, how initiators help specify replication origins at the molecular level and how their binding sites are established in higher eukaryotes remain foremost and long-standing questions in the field. This research project focuses on uncovering the molecular and structural principles for chromosomal binding site selection by the eukaryotic initiator, the origin recognition complex (ORC), in metazoan systems. Employing integrated biochemical, structural, and cell-based approaches, we aim to 1) elucidate how ORC binds DNA and how DNA structural elements contribute to this interaction, 2) determine how nucleosomes are recognized by ORC, and 3) identify auxiliary binding partners of ORC and establish how they contribute to origin specification.
Our main focus for the current funding period has been to understand how metazoan ORC engages DNA to elucidate the physical basis for the distinct DNA binding modes of eukaryotic initiators and how these properties help ORC discriminate between different accessible DNA regions across eukaryotic genomes. Using single-particle cryo-electron microscopy (cryo-EM), we have determined structures of various metazoan ORC-DNA assemblies at near-atomic resolution. These structures provide detailed insights into the molecular contacts between metazoan ORC and DNA and explain the different DNA sequence requirements for budding yeast and metazoan ORC. In addition, our structures identified a novel structural element in ORC that helps sense the DNA binding status of the initiator and transmits this information to ORC’s ATPase site, providing a mechanism for DNA-induced inhibition of ATP hydrolysis. In conjunction with biochemical efforts, our structural findings also suggest models for how DNA malleability may contribute to establishing sites for loading of replicative helicases onto DNA.
Our work to date has provided novel insights into the mechanisms of origin recognition in higher eukaryotes and of ORC-mediated replicative helicase loading. The outcomes of these and future efforts will set the foundation to understand at the molecular level how the replication initiation program is altered during cell differentiation and development. Our studies are also of biomedical relevance, as failure to precisely replicate chromosomal DNA leads to genetic instability, which in turn underpins many human diseases, including cancer and certain developmental disorders.