Biology subjects

Stewart, J. A.

Publications and source records attributed to Stewart, J. A..

8 recordsLinked to original sources

bioRxiv2026

High-fidelity rare structural variant detection with HiFiRE3 reduced representation via restriction enzyme ends

High-fidelity detection of rare structural variants (SVs) remains challenging because library preparation and sequencing techniques generate artifactual junctions that obscure true single-molecule events. Here, we present HiFiRe3, an error-minimized sequencing framework that combines artifact-aware library design with error suppression and correction strategies to enable rare SV detection and frequency assessment across long and short-read sequencing platforms. We first systematically characterized major classes of SV artifacts, including chimeric PCR products, intermolecular ligation, sequencing platform-specific artifacts, and mapping errors. HiFiRe3 supports error correction of these artifact junctions by combining reduced representation restriction fragments with pre-ligation size selection to enable computational filtering via independent forced restriction enzyme end (FREE) and <1N size logics. In nanopore libraries, these approaches enabled targeted detection of single-molecule SVs at replication stress hotspots in cultured human cells exposed to genotoxicants and in long genes in untreated mouse brains, while markedly reducing singleton translocation artifacts. HiFiRe3 extends to PacBio sequencing for joint SV and SNV error correction and to short-read platforms for cost-efficient high-fidelity nonhomologous SV analysis. Together, HiFiRe3 is a flexible framework for accurately detecting rare genomic structural variation with broad applicability to targeted and genome-wide studies by selective application of its error correction approaches.

genomics↗

bioRxiv2026

Mechanical tuning of replication stress tolerance and genomic stability through the checkpoint mediator Mrc1

Mrc1 is a replication fork component that mediates communication between the replication checkpoint and fork progression. Here, we find that Mrc1 forms a dynamic mechanical linkage between CMG helicase and DNA Polymerase {varepsilon} that balances fork stability and flexibility. In its coupled state, Mrc1 bridges CMG helicase and the Pol2 C-terminal domain of Polymerase {varepsilon} to maintain helicase-polymerase coordination, promoting tolerance to replication stress but constraining fork remodeling. Upon checkpoint activation, Mec1/Rad53-dependent phosphorylation of Mrc1 shifts this equilibrium toward a loosened state, reducing coupling and enabling fork flexibility and lower mutation rate at the cost of reduced stress resistance.

biochemistry↗

bioRxiv2025

Conditional deletion of human STN1 leads to telomere dysfunction and telomerase-dependent genome instability and proliferation defects

CTC1-STN1-TEN1 (CST) is a heterotrimeric, RPA-like complex that binds single-stranded DNA, stimulates DNA polymerase -primase, and functions in several genome maintenance pathways, including telomere maintenance and DNA replication/repair. During telomere replication, CST prevents telomerase from overextending the G-rich single-stranded overhang (G-OH) and promotes fill-in of the C-rich strand by stimulating DNA polymerase -primase. Previous work characterized the effects of CST loss by deleting CTC1 or TEN1. Interestingly, CTC1 knockout (KO) caused severe proliferation defects and telomeric damage signaling, whereas these phenotypes were absent following TEN1 KO. Molecular analysis revealed that, while loss of CTC1 or TEN1 leads to defective C-strand fill-in, only CTC1 KO exhibited excessive G-OH lengthening. Here, we characterized conditional STN1 KO cells and determined that STN1 KO leads to proliferation defects and telomeric damage signaling. Moreover, STN1 KO caused genome instability in the form of anaphase bridges and micronuclei. Interestingly, these phenotypes and growth inhibition were largely dependent on telomerase activity. Our findings indicate that STN1 KO closely resembles CTC1 versus TEN1 KO and that excessive G-OH extension underlies the genome instability caused by STN1 deletion. SUMMARY STATEMENTThe STN1 subunit of the single-stranded DNA binding protein CST prevents telomeric damage signaling, genome instability, and proliferation defects by limiting telomerase activity.

cell biology↗

bioRxiv2025

Formation of chromosomal rearrangements in Saccharomyces cerevisiae diploids through regionally-biased non-allelic homologous recombination

In earlier studies, we optimized an assay system for the genome-wide detection of copy number variation (CNV) in diploid Saccharomyces cerevisiae cells, based on selection for formaldehyde plus copper (FA+Cu) resistance conferred by the amplification of a dosage-dependent reporter cassette, SFA1-CUP1. Our analyses identified a robust bias for terminal deletions of the right arm of Chr7 (Chr7R) associated with unbalanced translocations. This bias was observed at approximately constant strength across all three sites where the amplification reporter cassette was inserted, in CNV-carrying yeast clones derived both spontaneously and from mutagen-induced recombinogenic conditions. We conducted allelic mitotic recombination experiments to investigate the possibility of the presence of a fragile site on Chr7R, but the results disfavored this model, and instead indicated that the Chr7R bias applies only to non-allelic rearrangements. We validated the existence of a CNV formation bias at Chr7R through an orthologous NAHR competition approach that was independent of selection for FA+Cu resistance. Finally, we showed the in contrast to its high participation in NAHR as a recipient sequence, Chr7R becomes amplified as a translocation donor less frequently than other comparable regions of the genome. To begin unraveling the cause of this unusual behavior, we evaluated the effect of a set of candidate genes involved in chromatin mobility and sister chromatid cohesion on the rearrangement spectra involving Chr7R. We found that deletion mutations in some of these genes, particularly SAP30, attenuated the biased NAHR behavior. Taken together, our results suggested that although Chr7R is not inherently more prone to DNA breakage than other regions, once a DNA lesion is formed there, it has a higher propensity to undergo inappropriate repair leading to a chromosomal rearrangement.

genetics↗

bioRxiv2025

Zone Matcher: A climate-based web application for deployment and assisted migration of forest trees

Populations of forest trees are generally adapted to the climates they inhabit. The farther trees are moved from their local climates, the more long-term growth and survival tend to decrease. Current tree deployment and assisted migration rely on climate distance thresholds (CDTs), which are climatic distances beyond which tree performance is considered unacceptable. Fixed zone systems, which have been used to guide deployment of native or orchard seedlots for more than 50 years, usually consist of contiguous geographic areas (zone units) divided into elevational bands (zones). In contrast, focal zone systems allow seed transfer among fixed zones that have similar climates. By using recent historical climates and future climate projections, focal zones can be used for current tree deployment or assisted migration. We developed a focal-zone system for the Pacific Northwest region of North America. First, we worked with stakeholders to select the base zones for the system. These consisted of geographic zone sets from Washington, Oregon, California, and Idaho/western Montana, and ecological zone sets from the U.S. and British Columbia. Second, by analyzing climate variation across the region, we developed a normalized Euclidean climate distance function consisting of nine climate variables from ClimateNA. Third, we inferred CDTs from analyses of climate variation within the base zones and from provenance tests. Fourth, we compared seed deployment areas using the fixed zone versus focal zone system, with and without assisted migration. Finally, we developed the Zone Matcher web application which implements our focal zone system. Across the region, we identified climate matches among 4,393 partially overlapping zones covering approximately 252 M ha. The unique area covered by these zones was about 167 M ha. Compared to fixed zones, our focal zone system increased the deployment area about 17-to 35-fold for the ecological zones and 70-to 300-fold for the geographic zones. This expands seed deployment options, allows more seedlots to be considered for a planting site, facilitates assisted migration, and simplifies sharing of seedlots among organizations. In addition to climate, seed transfer should also consider factors such as plantation soils, microtopography, and projections of competing vegetation, insects, diseases, and fire.

ecology↗

bioRxiv2024

Progerin Can Induce DNA Damage in the Absence of Global Changes in Replication or Cell Proliferation

Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare genetic condition characterized by features of accelerated aging, and individuals with HGPS seldom live beyond their mid-teens. The syndrome is commonly caused by a point mutation in the LMNA gene which codes for lamin A and its splice variant lamin C, components of the nuclear lamina. The mutation causing HGPS leads to production of a truncated, farnesylated form of lamin A referred to as "progerin." Progerin is also expressed at low levels in healthy individuals and appears to play a role in normal aging. HGPS is associated with an accumulation of genomic DNA double-strand breaks (DSBs) and alterations in the nature of DSB repair. The source of DSBs in HGPS is often attributed to stalling and subsequent collapse of replication forks in conjunction with faulty recruitment of repair factors to damage sites. In this work, we used a model system involving immortalized human cell lines to investigate progerin-induced genomic damage. Using an immunofluorescence approach to visualize phosphorylated histone H2AX foci which mark sites of genomic damage, we report that cells engineered to express progerin displayed a significant elevation of endogenous damage in the absence of any change in the cell cycle profile or doubling time of cells. Genomic damage was enhanced and persistent in progerin-expressing cells treated with hydroxyurea. Overexpression of wild-type lamin A did not elicit the outcomes associated with progerin expression. Our results show that DNA damage caused by progerin can occur independently from global changes in replication or cell proliferation.

molecular biology↗

bioRxiv2021

CST interacts with the cohesin complex and promotes chromosome cohesion

Sister chromatid cohesion (SCC) is established during DNA replication by loading of the cohesin complex on newly replicated chromatids. Cohesin must then be maintained until mitosis to prevent segregation defects and aneuploidy. How SCC is established and maintained until mitosis remains incompletely understood and emerging evidence suggests that replication stress can lead to premature SCC loss. Here, we report that the single-stranded DNA-binding protein CTC1-STN1-TEN1 (CST) aids in SCC. CST primarily functions in telomere length regulation but also has known roles in replication restart and DNA repair. Following depletion of CST subunits, we observed an increase in the complete loss of SCC. Additionally, we determined that CST interacts with the cohesin complex. Unexpectedly, we did not find evidence of defective cohesion establishment or mitotic progression in the absence of CST. However, we did find that treatment with various replication inhibitors increased the association between CST and cohesin. Since replication stress was recently shown to induce SCC loss, we supposed that CST may be required to maintain SCC following fork stalling. In agreement with this idea, SCC loss was greatly increased in CST-depleted cells following exogenous replication stress. Based on our findings, we propose that CST aids in the maintenance of SCC at stalled replication forks to prevent premature cohesion loss.

molecular biology↗

bioRxiv2020

Human CTC1 primarily functions in telomere maintenance/protection and promotes CHK1 phosphorylation in response to global replication stress

CST (CTC1-STN1-TEN1) is a heterotrimeric, RPA-like protein that binds to single stranded DNA (ssDNA) and functions in the replication of telomeric and non-telomeric DNA. Previous studies have shown that deletion of CTC1 results in decreased cell proliferation and telomeric DNA damage signaling. However, a detailed analysis of the consequences of conditional CTC1 knockout (KO) have not been fully elucidated. Here, we investigated the effects of CTC1 KO on cell cycle progression, genome-wide replication and activation of the DNA damage response. We find that CTC1 KO results in p53-mediated G2 arrest and increased apoptosis, but not genome-wide replication defects or DNA damage. Instead, the G2 arrest is dependent on the accumulation of telomeric RPA following CTC1 KO, suggesting that the primary function of CST is in telomere end protection and maintenance not genome-wide replication. However, despite increased RPA-ssDNA, global CHK1 phosphorylation was not detected in CTC1 KO cells. Further analysis revealed that CTC1 KO significantly inhibits CHK1 phosphorylation following hydroxyurea-induced replication stress, due to decreased levels of the ATR activator TopBP1. Overall, our results identify that telomere not genome-wide DNA damaging signaling leads to decrease proliferation following CTC1 deletion and that CST promotes ATR-CHK1 signaling through the regulation of TopBP1.

molecular biology↗