Biology subjects

Stillman, B.

Publications and source records attributed to Stillman, B..

9 recordsLinked to original sources

bioRxiv2026

Specification of Origins of DNA Replication Within Distinct Chromatin States in Human Cells

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{middle dot}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.

biochemistry↗

bioRxiv2026

A role for CIZ1, a Nuclear Matrix Protein Overexpressed in Cancer Cells, in Controlling the Initiation of DNA Replication

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.

molecular biology↗

bioRxiv2026

Evolution of Origin Sequence and Recognition for Licensing of Eukaryotic DNA Replication

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 lack 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.

molecular biology↗

bioRxiv2025

Genome sequence assembly and annotation of MATA and MATB strains of Yarrowia lipolytica

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.

genomics↗

bioRxiv2021

Prevalent and Dynamic Binding of the Cell Cycle Checkpoint Kinase Rad53 to Gene Promoters

Replication of the genome must be coordinated with gene transcription and cellular metabolism, especially following replication stress in the presence of limiting deoxyribonucleotides. The S. cerevisiae Rad53 (CHEK2 in mammals) checkpoint kinase plays a major role in cellular responses to DNA replication stress. Cell cycle regulated, genome-wide binding of Rad53 to chromatin was examined. Under replication stress, the kinase bound to sites of active DNA replication initiation and fork progression, but unexpectedly to the promoters of about 20% of genes encoding proteins involved in multiple cellular functions. Rad53 promoter binding correlated with changes in expression of a subset of genes. Rad53 promoter binding to certain genes was influenced by sequence-specific transcription factors and less by checkpoint signaling. However, in checkpoint mutants, untimely activation of late-replicating origins reduces the transcription of nearby genes, with concomitant localization of Rad53 to their gene bodies. We suggest that the Rad53 checkpoint kinase coordinates genome-wide replication and transcription under replication stress conditions.

molecular biology↗

bioRxiv2020

The dynamic nature of the human Origin Recognition Complex revealed through five cryoEM structures

Genome replication is initiated from specific origin sites established by dynamic events. The Origin Recognition Complex (ORC) is necessary for orchestrating the initiation process by binding to origin DNA, recruiting CDC6, and assembling the MCM replicative helicase on DNA. Here we report five cryoEM structures of the human ORC (HsORC) that illustrate the native flexibility of the complex. The absence of ORC1 revealed a compact, stable complex of ORC2-5. Introduction of ORC1 opens the complex into several dynamic conformations. Two structures revealed dynamic movements of the ORC1 AAA+ and ORC2 winged-helix domains that likely impact DNA incorporation into the ORC core. Additional twist and pinch motions were observed in an open ORC conformation revealing a hinge at the ORC5{middle dot}3 interface that may facilitate ORC binding to DNA. Finally, a structure of ORC was determined with endogenous DNA bound in the core revealing important differences between human and yeast origin recognition.

biochemistry↗

bioRxiv2020

The human Origin Recognition Complex is essential for pre-RC assembly, mitosis and maintenance of nuclear structure.

The Origin Recognition Complex (ORC) cooperates with CDC6, MCM2-7, and CDT1 to form pre- RC complexes at origins of DNA replication. Here we report tiling-sgRNA CRISPR screens that show that each subunit of ORC and CDC6 are essential in human cells. Using an auxin-inducible degradation system, stable cell lines were created that ablate ORC2 rapidly, revealing multiple cell division cycle phenotypes. The primary defect in the absence of ORC2 was cells encountering difficulty in initiating DNA replication or progressing through the cell division cycle due to reduced MCM2-7 loading onto chromatin in G1 phase. The nuclei of ORC2 deficient cells were also large, with decompacted heterochromatin. Some ORC2 deficient cells that completed DNA replication entered into, but never exited mitosis. ORC1 knockout cells also demonstrated extremely slow cell proliferation and abnormal cell and nuclear morphology. Thus, ORC proteins and CDC6 are indispensable for normal cellular proliferation and contribute to nuclear organization.

molecular biology↗

bioRxiv2020

Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

DNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.Competing Interest StatementThe authors have declared no competing interest.View Full Text

molecular biology↗

bioRxiv2019

Cyclin binding Cy motifs have multiple activities in the initiation of DNA replication

The initiation of DNA replication involves the cell cycle-dependent assembly and disassembly of protein complexes, including the Origin Recognition Complex (ORC) and CDC6 AAA+ ATPases. We report that multiple short, linear protein motifs (SLiMs) within intrinsically disordered regions in ORC1 and CDC6, including Cyclin-binding (Cy) motifs, mediate Cyclin-CDK dependent and independent protein-protein interactions, conditional on cell cycle phase. The ORC1 Cy motif mediates an auto-regulatory self-interaction, and the same Cy motif prevents CDC6 binding to ORC1 in mitosis, but then facilitates the destruction of ORC1 in S phase. In contrast, in G1, the CDC6 Cy motif promotes ORC1-CDC6 interaction independent of Cyclin-CDK protein phosphorylation. CDC6 interaction with ORC also requires a basic region of ORC1 that in yeast mediates ORC-DNA interactions. We also demonstrate that protein phosphatase 1 binds directly to a SLiM in ORC1, causing de-phosphorylation upon mitotic exit. Thus, Cy-motifs have wider roles, functioning as a ligand and as a degron.

molecular biology↗