Bruce Stillman

A comprehensive map of binding sites for the Origin Recognition Complex (ORC) in the human genome reveals relationships among replication licensing, chromatin state, three-dimensional genome organization, location of origins of DNA replication and DNA replication timing. Defining these relationships is essential for understanding how replication licensing is integrated with the broader organization and duplication of the human genome. Using 30 high-quality ORC1 CUT&RUN datasets, an ORC1 three-dimensional binding matrix (ORC1 3DM) was constructed that captures genomic position, binding intensity, and reproducibility across replicates. This analysis reveals considerable heterogeneity in ORC1 binding, and integration with chromatin-state maps shows that ORC1-binding sites separate into distinct epigenomic groups, with high-confidence sites enriched in H2A.Z1- and H3K4me3-associated open chromatin. Hi-C analysis demonstrates preferential localization of ORC1 binding to sub-regions of genomic A compartments and frequent association with loop anchors, supported by a direct interaction between ORC1 and CTCF. Replication-timing analysis shows that ORC1-binding sites are enriched in initiation zones and exhibit subgroup-specific temporal patterns. An integrated analysis combining multiple replication initiation assays and chromatin features further places ORC1 3DM within a unified replication–chromatin framework. Together, these results support a hierarchical, probabilistic model of replication licensing that is embedded within chromatin architecture. Significance Statement: Understanding how DNA replication licensing relates to chromatin organization requires quantitative mapping and integration of genomic features. We developed an ORC1 three-dimensional binding matrix (ORC1 3DM) from 30 CUT&RUN datasets, quantifying genomic binding coordinates, intensity and reproducibility. We established an integrative framework to compare diverse genomic datasets across 25 chromatin states and a high-resolution approach to characterize replication timing profiles and their relationships with replication initiation. These approaches revealed distinct chromatin-associated ORC1-binding groups, preferential localization of high-confidence ORC1 sites in active chromatin and highly interactive A-compartment subregions, and subgroup-specific relationships with replication initiation and timing. Together, these findings and methodological advances provide a quantitative framework for investigating relationships among replication licensing, chromatin state, three-dimensional genome organization, and replication timing.

DNA replication licensing is tightly coordinated with cell-cycle progression to ensure genome stability. Here we demonstrate that CIZ1, a nuclear matrix scaffold protein in human cells, coordinates DNA replication licensing through direct interactions with the Origin Recognition Complex (ORC), CDC6, and Cyclins E/A–CDK2. The C-terminal zinc finger region of CIZ1 mediates binding to the cyclin-binding motifs of ORC1 and CDC6, and Cyclin–CDK-dependent phosphorylation regulates these interactions. Cell cycle analyses demonstrated a dynamic association of CIZ1 with ORC1 and CDC6. Functional studies showed that CIZ1 depletion impaired MCM loading, reduced DNA synthesis, delayed S-phase progression and inhibited cell proliferation. Cancer cells overexpressing CIZ1 exhibited an enhanced dependency on its expression, and CRISPR tiling screens identified the C-terminal regulatory region containing cyclin-binding motifs and CDK phosphorylation sites as essential for cell proliferation. Acute degradation of CIZ1 further revealed an unexpected role in maintaining CCCTC-binding factor (CTCF) occupancy at specific genomic loci and regulating transcriptional programs associated with cell adhesion and migration, indicating that CIZ1 links DNA replication with higher-order genome organization. Finally, depletion of CIZ1 significantly suppressed colorectal tumor growth in xenograft models. Together, these findings establish CIZ1 as a multifunctional scaffold that integrates DNA replication licensing, Cyclin– CDK signaling, and chromatin architecture.

Pediatric sarcomas are a heterogeneous group of rare mesodermal malignancies. These cancers, which affect children from infancy through adolescence and young adulthood, are in general challenging to treat with currently available therapies. Biologically, many are characterized by quiet genomes, fusion oncoproteins, immune “cold” microenvironments, and vast epigenetic deregulation that contributes to diverse and complex mechanistic drivers. Multifaceted advancements in research strategies, including high-throughput screening, new model systems, surfaceome profiling, and study of oncogenic fusion condensates have led to new opportunities for understanding the biology of pediatric sarcomas. To continue to make progress for these difficult to treat cancers, it will be critical to continue to improve access to bioinformatic data, approach patient care using innovative clinical trial frameworks, and foster interdisciplinary partnerships among medicinal chemists, scientists, clinicians, advocates, and industry partners.

The large size of eukaryotic chromosomes necessitates that the initiation of DNA replication occurs at numerous origins of DNA replication. In S. cerevisiae , origins are highly DNA sequence-specific and are recognized by the Origin Recognition Complex (ORC). In contrast, most eukaryotes have lost features in ORC subunits that contribute to DNA sequence-specific recognition, raising the question of how origins are identified. An analysis of origins in the genome of the distantly related budding yeast Yarrowia lipolytica identified considerable variability in origin sequence and structure. High-resolution structures demonstrate that Y. lipolytica origins are recognized by a combination of ORC and Cdc6 in a manner different from S. cerevisiae . The structure of Yarrowia ORC-Cdc6 bound to different origins shows considerable plasticity in protein-DNA interactions. We compare these yeast structures to the structure of human ORC-CDC6 bound to DNA. These studies reveal information on the evolution of origins and origin recognition. Nomenclature note: There is a different nomenclature for proteins in yeast and human cells. For example, Cdc6 in yeasts is CDC6 in human cells.

James Dewey Watson who died on November 7, 2025, at the age of 97, was a towering figure in 20th century life science. With Francis Crick, he determined the structure of DNA, laying the foundation of modern biology; he made Cold Spring Harbor Laboratory one of the world’s great biomedical research institutes and a center for professional education in molecular biology and neuroscience; he established the Human Genome Project and ensured that research on the associated ethical, legal, and social issues was part of the project; he created a classic textbook that revolutionized the style of the genre; and he wrote one of the most interesting and controversial books on science for scientists and nonscientists alike.

Yeast is commonly utilized in molecular and cell biology research, and Yarrowia lipolytica is favored by bioengineers due to its ability to produce copious amounts of lipids, chemicals, and enzymes for industrial applications. Y. lipolytica is a dimorphic yeast that can proliferate in aerobic and hydrophobic environments conducive to industrial use. However, there is limited knowledge about the basic molecular biology of this yeast, including how the genome is duplicated and how gene silencing occurs. Genome sequences of Y. lipolytica strains have offered insights into this yeast species and have facilitated the development of new industrial applications. Although previous studies have reported the genome sequence of a few Y. lipolytica strains, it is of value to have more precise sequences and annotation, particularly for studies of the biology of this yeast. To further study and characterize the molecular biology of this microorganism, a high-quality reference genome assembly and annotation has been produced for two related Y. lipolytica strains of the opposite mating type, strain E122 (MATA) and 22301-5 (MATB). The combination of short-read and long-read sequencing of genome DNA and short-read and long-read sequencing of transcript cDNAs allowed the genome assembly and a comparison with a distantly related Yarrowia strain.

The initiation of DNA replication in eukaryotic cells begins with the assembly of pre-replicative complexes (pre-RCs) at many sites along each chromosome during the G1 phase of the cell cycle. Pre-RCs license each chromosome for duplication during S phase and mark the origins of DNA replication. In this Review, we discuss and contextualize recent findings identifying the mechanisms of origin recognition and pre-RC assembly mediated by the origin recognition complex (ORC), Cdc6 and the Mcm2–Mcm7 (Mcm2-7) hexamer bound to Cdt1. We also present comprehensive videos that demonstrate the multiple mechanisms for pre-RC assembly and compare the structures of the complexes involved in human and Saccharomyces cerevisiae cells.

Yeast is commonly utilized in molecular and cell biology research, and Yarrowia lipolytica is favored by bio-engineers due to its ability to produce copious amounts of lipids, chemicals, and enzymes for industrial applications. Y. lipolytica is a dimorphic yeast that can proliferate in aerobic and hydrophobic environments conducive to industrial use. However, there is limited knowledge about the basic molecular biology of this yeast, including how the genome is duplicated and how gene silencing occurs. Genome sequences of Y. lipolytica strains have offered insights into this yeast species and have facilitated the development of new industrial applications. Although previous studies have reported the genome sequence of a few Y. lipolytica strains, it is of value to have more precise sequences and annotation, particularly for studies of the biology of this yeast. To further study and characterize the molecular biology of this microorganism, a high-quality reference genome assembly and annotation has been produced for two related Y. lipolytica strains of the opposite mating type, strain E122 ( MATA ) and 22301-5 ( MATB ). The combination of short-read and long-read sequencing of genome DNA and short-read and long-read sequencing of transcript cDNAs allowed the genome assembly and a comparison with a distantly related Yarrowia strain.

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC . This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria.