Biology subjects

Cameron, D. P.

Publications and source records attributed to Cameron, D. P..

4 recordsLinked to original sources

bioRxiv2025

MYC modulates TOP2A diffusion to promote substrate detection and activity

Topoisomerases alleviate DNA supercoiling by cleaving and resealing DNA strands. Previously, we showed that the oncoprotein MYC recruits and stimulates topoisomerases to remove DNA entanglements generated by oncogenic transcription. Understanding this mechanism may suggest methods to inhibit MYC-driven topoisomerase activation, targeting tumor-specific transcription. Here, we demonstrate that the essential topoisomerase TOP2A in human cells exists in a dynamic equilibrium between sequestration in the nucleolus, substrate searching in transcription hubs, and active engagement on chromatin. This equilibrium is highly responsive to changes in DNA topology, allowing cells to regulate TOP2A levels. Using single molecule tracking, we show that MYC accelerates TOP2A diffusion in cells. We explain this phenotype by demonstrating that MYC limits TOP2A self-interaction in vitro, while decreasing the size of TOP2A complexes in cells. By increasing TOP2A diffusion, MYC promotes substrate binding and increases TOP2A engagement on chromatin genome-wide, revealing the mechanism underlying MYC stimulation of TOP2A activity.

cell biology↗

bioRxiv2023

Co-inhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills pancreatic cancer via readthrough transcription

Pancreatic carcinoma is one of the most lethal cancers and the absence of efficient therapeutic strategies results in poor prognosis. Transcriptional dysregulation due to alterations in KRAS and MYC impacts initiation, development, and survival of this tumor type. Using patient-derived xenografts of pancreatic carcinoma driven by KRAS and MYC oncogenic transcription, we show that co-inhibition of Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) synergistically induce tumor regression through targeting promoter pause-release, a rate-limiting step in transcription elongation. By comparing the nascent transcriptome with the recruitment of elongation and termination factors along genes, we found that co-inhibition of TOP1 and BRD4, while globally impairing RNA production, disturbs recruitment of proteins involved in termination. Thus, RNA polymerases continue transcribing downstream of genes for hundreds of kilobases leading to readthrough transcription. This pervasive transcription also occurs during replication, perturbing replisome progression and leading to DNA damage. The synergistic effect of TOP1 and BRD4 inhibition is specific for cancer cells leaving normal cells unharmed, highlighting the sensitivity of the tumor to these transcriptional defects. This preclinical study provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors to treat pancreatic carcinomas addicted to oncogenic drivers of high transcription and replication. One Sentence SummaryTOP1 and BRD4 inhibitors synergize to selectively kill pancreatic cancer in vivo via readthrough transcription without emergence of drug resistance

cancer biology↗

bioRxiv2021

The Epstein-Barr virus ubiquitin deconjugase BPLF1 regulates the activity of Topoisomerase II during virus replication

Topoisomerases are essential for the replication of herpesviruses but the mechanisms by which the viruses hijack the cellular enzymes are largely unknown. We found that topoisomerase-II (TOP2) is a substrate of the Epstein-Barr virus (EBV) ubiquitin deconjugase BPLF1. BPLF1 selectively inhibited the ubiquitination of TOP2 following treatment with topoisomerase poisons, interacted with TOP2 and TOP2{beta} in co-immunoprecipitation and in vitro pull-down, stabilized Etoposide-trapped TOP2 cleavage complexes (TOP2cc) and promoted TOP2 SUMOylation, which halted the DNA-damage response and reduced Etoposide toxicity. Induction of the productive virus cycle promoted the accumulation of TOP2{beta}cc, enhanced TOP2{beta} SUMOylation, and reduced Etoposide toxicity in lymphoblastoid cell lines carrying recombinant EBV encoding the active enzyme. Attenuation of this phenotype upon expression of a catalytic mutant BPLF1-C61A impaired viral DNA synthesis and virus release. These findings highlight a previously unrecognized function of BPLF1 in promoting non-proteolytic pathways for TOP2cc debulking that favor cell survival and virus production.

microbiology↗

bioRxiv2020

Optimal and regulated transcription facilitates formation of damage-induced cohesion

The structural maintenance of chromosome (SMC) complex cohesin mediates sister chromatid cohesion established during replication, and damage-induced cohesion formed in response to DSBs post-replication. The translesion synthesis polymerase Pol{eta} is required for damage-induced cohesion through a hitherto unknown mechanism. Since Pol{eta} is functionally associated with transcription, and transcription triggers de novo cohesion in Schizosaccharomyces pombe, we hypothesized that transcription facilitates damage-induced cohesion in Saccharomyces cerevisiae. Here, we show dysregulated transcriptional profiles in Pol{eta}-depleted cells (rad30{Delta}), where genes involved in chromatin assembly and positive transcription regulation were downregulated. In addition, chromatin association of RNA polymerase II was reduced at promoters and coding regions in rad30{Delta} compared to WT cells, while occupancy of the H2A.Z variant (Htz1) at promoters was increased in rad30{Delta} cells. Perturbing histone exchange at promoters inactivated damage-induced cohesion, similarly to deletion of the RAD30 gene. Conversely, altering regulation of transcription elongation suppressed the deficient damage-induced cohesion in rad30{Delta} cells. These results indicate that Pol{eta} has an assisting role during the transcriptional process, which consecutively facilitates formation of damage-induced cohesion. This further suggests a potential linkage between regulation of transcription and formation of damage-induced cohesion after replication. Author SummaryThe cohesin complex dynamically associates with chromosomes and holds sister chromatids together through cohesion established during replication. This ensures faithful chromosome segregation at anaphase. In budding yeast, DNA double strand breaks trigger sister chromatid cohesion even after replication. This so-called damage-induced cohesion is formed both close to the breaks, and genome-wide on undamaged chromosomes. The translesion synthesis polymerase eta (Pol{eta}) is specifically required for genome wide damage-induced cohesion. Although Pol{eta} is well characterized for its function in bypassing ultraviolet-induced DNA lesions, its mechanistic role in damage-induced cohesion is unclear. Here, we show that transcriptional regulation is perturbed in the absence of Pol{eta}. We propose that Pol{eta} could aid in chromatin association of RNA polymerase II through phosphorylation of the Pol{eta}-S14 residue, a non-canonical role of Pol{eta} which further facilitates formation of damage-induced cohesion genome wide. In addition, we observe the need of replication-independent nucleosome assembly/histone exchange for formation of damage-induced cohesion. This together provides new insight into formation of damage-induced cohesion after replication, which will be interesting to further explore.

cell biology↗