Biology subjects

Sansam, C. L.

Publications and source records attributed to Sansam, C. L..

6 recordsLinked to original sources

bioRxiv2026

Adaptive Replication Fork Acceleration by CDK1-Cyclin B1 Sustains Genome Duplication despite Impaired Origin Firing

Although MTBP is essential for replication origin firing, we show here that strong depletion of MTBP can have minor effects on DNA replication rates. This suggests an adaptive process in the DNA replication program, so we examined mechanisms underlying this plasticity. Using an auxin-inducible degron to deplete MTBP, we found that acute suppression of MTBP blocked DNA replication, but that replication rates recovered over time. The timing of this recovery paralleled S phase expression of Cyclin B1, and inhibition of CDK1-Cyclin B1 prevented the recovery. Recovery did not involve restoration of origin firing; instead, replication recovered through accelerated fork progression. Consistent with CDK1 driving this acceleration, ATR inhibition, which activates CDK1, stimulated DNA replication in MTBP-depleted cells through CDK1-dependent increased fork progression rather than increased origin firing. Knockdown of RIF1, a known CDK1 target, phenocopied this effect. Although RIF1 is best known for opposing DDK-dependent MCM phosphorylation at origins, we find that RIF1 knockdown stimulates replication even when DDK is inhibited. Furthermore, RIF1 loss increased replication by accelerating fork progression rather than increasing origin firing. Together, these findings reveal a CDK1-RIF1-dependent mechanism that promotes fork speed during S phase and defines a form of replication plasticity in which fork rate compensates for reduced origin firing. SIGNIFICANCE STATEMENTAccurate genome duplication requires thousands of replication origins to fire and replication forks to complete DNA synthesis on schedule. When origin firing is compromised, it is unclear how cells avoid replication failure. We show that cells adapt to persistent loss of the origin-firing factor MTBP by accelerating replication fork progression through a CDK1-RIF1-dependent mechanism, partially compensating for reduced initiation. This adaptive response defines a form of replication plasticity in which cells rebalance origin usage and fork speed to sustain DNA synthesis. This mechanism may be especially relevant in cancer cells or other contexts where replication initiation is chronically stressed.

molecular biology↗

bioRxiv2025

DUE-B Is Dispensable for Early Development and Genome Duplication in Vertebrates.

The DNA Unwinding Element-Binding protein (DUE-B) is a Cyclin Dependent Kinase (CDK) and Dbf4-Dependent Kinase (DDK) substrate that has been implicated in the control of DNA replication initiation. Previous studies reported that knocking down DUE-B in HeLa cells perturbs the G1-to-S phase transition, while depleting DUE-B from interphase Xenopus egg extracts impairs replication initiation. Based on these findings, the prevailing view is that DUE-B is a vertebrate-specific DNA replication initiation factor. Here, we asked whether due-b was an essential vertebrate gene in vivo, and whether it was critical for proper embryonic development in the zebrafish Danio rerio. We have generated due-b mutant zebrafish through genome-editing TALENs that fail to express due-b mRNA or protein. These mutant zebrafish are viable and survive to adulthood. They do not display outward developmental phenotypes, and when stressed with replication inhibitors, do not differ from their wild-type counterparts. Cell cycle analysis demonstrates that DNA replication occurs normally. Consistent with the zebrafish data, immunodepleting DUE-B from Xenopus nuclear egg extract did not impair DNA replication. Taken together, our findings indicate that DUE-B is dispensable for DNA replication and early development in vertebrates. SummaryThe DNA replication factor DUE-B is not required for zebrafish development or genome duplication, suggesting it plays a redundant or specialized role in DNA replication.

developmental biology↗

bioRxiv2025

Dynamic regulation of origin firing factors links CDK activity to dormant origin activation

Dormant replication origins help ensure complete genome duplication when replication forks stall, yet how these origins are activated remains poorly understood. Here, we identify a novel regulatory mechanism by which cyclin-dependent kinase (CDK) activity controls the abundance and chromatin recruitment of the origin firing factors TRESLIN and MTBP to promote dormant origin activation. Inhibition of WEE1 kinase during S phase increases CDK activity, which blocks the PCNA-dependent degradation of TRESLIN and enhances its chromatin association along with MTBP. This increased loading is required for elevated helicase recruitment and DNA synthesis under CDK-hyperactive conditions. These effects are reversed by CDK inhibition and depend on both TRESLIN and MTBP. We define a conserved sequence within TRESLIN required for its CDK-sensitive degradation. Significantly, the recruitment of TRESLIN-MTBP and loading of helicase exceed levels observed in unperturbed S phase, supporting a model in which dormant origin firing is actively upregulated through CDK-mediated stabilization of the initiation machinery. These findings uncover a new control point in replication origin usage with implications for genome stability and therapeutic kinase inhibition.

cell biology↗

bioRxiv2024

Cell Cycle-Dependent TICRR/TRESLIN and MTBP Chromatin Binding Mechanisms and Patterns

The selection of replication origins is a defining characteristic of DNA replication in eukaryotes, yet its mechanism in humans has not been well-defined. In this study, we use Cut&Run to examine genomic binding locations for TICRR/TRESLIN and MTBP, the human orthologs for the yeast DNA replication initiation factors Sld3 and Sld7. We mapped TRESLIN and MTBP binding in HCT116 colorectal cancer cells using asynchronous and G1 synchronized populations. Our data show that TRESLIN and MTBP binding patterns are more defined in a G1 synchronized population compared to asynchronously cycling cells. We also examined whether TRESLIN and MTBP are dependent on one another for binding. Our data suggest MTBP is dependent on TRESLIN for proper association with chromatin during G1 but not S phase. Finally, we asked whether TRESLIN and MTBP binding to chromatin requires licensed origins. Using cell lines with a non-degradable inducible Geminin to inhibit licensing, we show TRESLIN and MTBP binding does not require loaded MCMs. Altogether, our Cut&Run data provides evidence for a chromatin binding mechanism of TRESLIN-MTBP during G1 that is dependent on TRESLIN and does not require interactions with licensed origins.

genomics↗

bioRxiv2022

Zebrafish Rif1 Impacts Zygotic Genome Activation, Replication Timing, and Sex Determination

Deregulated DNA replication causes human developmental disorders and cancer, but we know little about how DNA replication is coordinated with changes in transcription and chromatin structure. The initiation of replication forks follows a spatiotemporal pattern called the replication timing program. We have developed the zebrafish into a model system to study the mechanisms by which the replication timing program changes during the extensive changes in the cell cycle, transcription, chromatin organization, and nuclear structure that occur during development. Our previous studies identified changes in DNA replication timing patterns occurring from the onset of zygotic transcription through gastrulation in zebrafish embryos. Rif1 is required for DNA replication timing in a wide range of eukaryotes. The broader role of Rif1 in establishing the replication timing program and chromatin structure during early vertebrate development remains unknown. We have generated Rif1 mutant zebrafish and have performed RNA sequencing and whole-genome replication timing analyses on multiple developmental stages. Rif1 mutants were viable but had a defect in female sex determination. Surprisingly, Rif1 loss predominantly affected DNA replication timing after gastrulation, while its impacts on transcription were more substantial during zygotic genome activation. Our results indicate that Rif1 has distinct roles in DNA replication and transcription control that manifest at different stages of development.

cell biology↗

bioRxiv2022

Dynamic Features of Chromosomal Instability during Culture of Induced Pluripotent Stem Cells

Induced pluripotent stem cells (iPSCs) hold great potential for regenerative medicine. By reprogramming a patient"s own cells, immunological rejection can be avoided during transplantation. For expansion and gene editing, iPSCs are grown in artificial culture for extended times. Culture affords potential danger for accumulation of genetic aberrations. To study these, two iPS cell lines were cultured and periodically analyzed using advanced optical mapping to detect and classify chromosome numerical and segmental changes that included deletions, insertions, balanced translocations and inversions. In one of the lines, a population trisomic for chromosome 12 gained dominance over a small number of passages. This appearance and dominance of the culture by chromosome 12 trisomic cells was tracked through intermediate passages by analysis of chromosome spreads. Mathematical modeling suggested that the proliferation rates of diploid versus trisomic cells could not account for the rapid dominance of the trisomic population. In addition, optical mapping revealed hundreds of structural variations distinct from those generally found within the human population. Many of these structural variants were detected in samples taken early in the culturing process and were maintained in late passage samples, while others were acquired over the course of culturing.

cell biology↗