Biology subjects

Ramsden, D. A.

Publications and source records attributed to Ramsden, D. A..

5 recordsLinked to original sources

bioRxiv2026

A RHNO1-ATR/Chk1 positive feedback loop sustains the DNA replication stress response

Elevated DNA replication stress is a common feature of cancer cells, rendering them dependent on ATR/Chk1 signaling, which controls the DNA replication stress checkpoint, for survival. Although the activation of ATR/Chk1 signaling is well-established, how this signal is maintained until replication stress is fully resolved is less understood. Here, we investigated the roles of RAD9-HUS1-RAD1 interacting nuclear orphan 1 (RHNO1) in cancer progression and its involvement in maintaining ATR/Chk1 signaling during the DNA replication stress response. We demonstrate that depletion of RHNO1 significantly inhibits cancer cell proliferation in vitro and tumor growth in vivo. Mechanistically, we show that, while RHNO1 is dispensable for initial ATR/Chk1 signal activation, it is upregulated and stabilized following replication stress. RHNO1 is required to sustain ATR/Chk1 signaling, prevent premature checkpoint collapse, and suppress genomic instability. Under basal conditions, RHNO1 is rapidly degraded by the proteasome, however, RHNO1 is phosphorylated and stabilized following DNA replication stress. Stabilization of RHNO1 is mediated by ATR/Chk1 signaling which promotes RHNO1 localization to stressed replication forks marked by phosphorylated RPA32. Together, our data reveals a novel positive feedback loop wherein ATR/Chk1 signaling activation stabilizes RHNO1, which, in turn, is required to sustain the signal and thus the replication stress response. This work identifies RHNO1 as a key component in the cellular replication stress response and highlights its potential as a therapeutic target for tumor cells reliant on ATR/Chk1 signaling.

cancer biology↗

bioRxiv2025

Hierarchical Coordination of Polymerase Theta and RAD51 Resolves Clustered Replication Fork Collapse

The essential role of polymerase theta (Pol{theta})-mediated end joining (TMEJ), an alternative double strand break repair pathway, has been primarily studied in homologous recombination (HR)-deficient contexts(1, 2). Here, we uncover an indispensable role for TMEJ in HR-proficient mammalian cells during the repair of interstrand crosslinks (ICLs). We show that Pol{theta} is recruited downstream of canonical ICL repair steps--including ICL unhooking, RAD51 loading, and RAD51 ubiquitylation--and localizes to sites of unresolved HR through interactions with ubiquitylated RAD51 filaments. Using genomic scar profiling and targeted ICL repair assays, we find that TMEJ resolves a minor subset of lesions that are not amenable to HR repair, such as clustered ICLs that can induce two-ended replication fork collapse. These findings reveal a RAD51 ubiquitylation-dependent mechanism for Pol{theta} recruitment and establish TMEJ as a hierarchically deployed DNA repair pathway that safeguards genome stability when HR is insufficient to resolve replication-associated DNA damage. Short SummaryPol{theta} is recruited via RAD51 ubiquitylation to resolve clustered ICLs that generate HR-refractory replication fork collapse.

cell biology↗

bioRxiv2025

BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance

Extrachromosomal circular DNA (ecDNA) are commonly produced within the nucleus to drive genome dynamics and heterogeneity, enabling cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes mediate ecDNA production. Following DNA fragmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively catalyzes ecDNA production in Drosophila and mammals, with patient tumor ecDNA harboring junctions marked by LIG4 activity. Notably, disrupting LIG4 or BRCA1-A in cancer cells impairs ecDNA-mediated adaptation, hindering resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers new therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

cancer biology↗

bioRxiv2020

DNA polymerase theta suppresses mitotic crossing over

Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol {theta}) to use resected, 3 single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we expand the number of homologous recombination proteins synthetic lethal with Pol {theta} to the Holliday junction resolvases SLX4 and GEN1. SLX4 and GEN1 are required for viability in the absence of Pol {theta} in Drosophila melanogaster, and lack of all three proteins results in very high levels of apoptosis. We observe that flies deficient in Pol {theta} and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol {theta} or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol {theta}. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol {theta} can thus compensate for the loss of the Holliday junction resolvases by utilizing homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells. Author summaryChromosome breaks are a common threat to the stability of DNA. Mutations in genes involved in the early steps of homologous recombination (BRCA1 and BRCA2), a mostly error-free chromosome break repair pathway, lead to hereditary breast cancer. Cells lacking BRCA1 and BRCA2 rely on DNA polymerase theta, a key protein for a more error-prone pathway, for survival. Using fruit flies and mammalian cells, we have shown that mutations in genes involved in later steps of homologous recombination (SLX4 and GEN1) also make cells reliant on polymerase theta. Moreover, we have shown that polymerase theta acts upstream of a type of homologous recombination that is error-prone and depends on SLX4 and GEN1. This form of homologous recombination, termed Holliday junction resolution, creates mitotic crossovers, which can lead to loss of heterozygosity and cancer. Our results expand the cellular contexts that make cells depend on polymerase theta for survival, and the substrates that this protein can use to repair chromosome breaks.

genetics↗

bioRxiv2019

Mechanistic Basis for Microhomology Identification and Genome Scarring by Polymerase Theta

DNA Polymerase Theta mediates an end joining pathway (TMEJ) that repairs chromosome breaks. It requires resection of broken ends to generate long, 3 single stranded DNA tails, annealing of complementary sequence segments (microhomologies) in these tails, followed by microhomology-primed synthesis sufficient to resolve broken ends. The means by which microhomologies are identified is thus a critical step in this pathway, but is not understood. Here we show microhomologies are identified by a scanning mechanism initiated from the 3 terminus and favoring bi-directional progression into flanking DNA, typically to a maximum of 15 nucleotides into each flank. Polymerase theta is frequently insufficiently processive to complete repair of breaks in microhomology-poor, AT-rich regions. Aborted synthesis leads to one or more additional rounds of microhomology search, annealing, and synthesis; this promotes complete repair in part because earlier rounds of synthesis generate microhomologies de novo that are sufficiently long that synthesis is more processive. Aborted rounds of synthesis are evident in characteristic genomic scars as insertions of 3-30 bp of sequence that is identical to flanking DNA ("templated" insertions). Templated insertions are present at higher levels in breast cancer genomes from patients with germline BRCA1/2 mutations, consistent with an addiction to TMEJ in these cancers. Our work thus describes the mechanism for microhomology identification, and shows both how it mitigates limitations implicit in the microhomology requirement, and generates distinctive genomic scars associated with pathogenic genome instability.

molecular biology↗