Biology subjects

Webster, S.

Publications and source records attributed to Webster, S..

8 recordsLinked to original sources

bioRxiv2025

Mutations and structural variants arising during double-strand break repair

Double-strand break (DSB) repair is highly mutagenic compared to normal replication. In budding yeast, repair of an HO endonuclease-induced DSB at MAT can be repaired by using a transcriptionally silent HMR::Kl-URA3 donor. During repair, -1 deletions in homonucleotide runs are strongly favored over +1 insertions, whereas during replication, spontaneous +1 and -1 events are equal. Microhomology-bounded, repair-associated intragenic deletions (IDs) are recovered 12 times more frequently than tandem duplications (TDs). IDs have a mean length of 56 bp, while TDs average 22 bp. These data suggest a picture of the structure of the repair replication fork: IDs and TDs occur within the open structure of a migrating D-loop, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either [~]80 bp ahead or [~]40 bp behind the 3 end. Another major class of repair-associated mutations ([~]10%) are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence in HMR is only 72% identical (homeologous) with S. cerevisiae ura3-52. ICTS events begin and end at regions of short ([~]7 bp) microhomology; however, ICTS events are constrained to the middle of the copied sequence. Whereas microhomology usage in intragenic deletions is not influenced by adjacent homeology, we show that extensive pairing of adjacent homeology plays a critical role in ICTS. Thus, although by convention, structural variants are characterized by the precise base pairs at their junction, microhomology-mediated template switching actually requires alignment of extensive adjacent homeology. Significance statementDNA synthesis during repair of a double-strand chromosome break by homologous recombination exhibits a high rate of mutation compared to normal replication. Using a genetic system in budding yeast, we isolated thousands of mutations occurring during repair. We conclude that the repair replication fork appears to have the two DNA strands open [~]80 bp ahead of the DNA polymerase, but the strands re-anneal rapidly behind the polymerase. Additionally, we analyzed interchromosomal template switching, in which the partially copied DNA strand dissociates and pairs with a new template at a short stretch of perfectly matching bases (microhomology), and resumes copying. We show that these apparent microhomology-mediated template switching events in fact require the pairing of [~]200 bp of imperfectly matching bases (homeology).

genetics↗

bioRxiv2024

Recurrent breakpoints in the BRD4 locus reduce toxicity associated with gene amplification

Structural variants (SVs) represent a mechanism by which cancer cells activate oncogenes or disrupt the function of genes with tumor suppressor roles. A recent study by the PCAWG Consortium investigated structural variants in 30 tumor types, identifying focal rearrangements in the oncogenic BRD4 gene in ovarian, endometrial and breast cancers. These rearrangements resulted in decreased BRD4 expression despite increased copy number, suggesting a novel mechanism to finetune gene over-expression. In this study, we show that focal deletions of BRD4 disrupt genomic regulatory regions and impact gene isoform expression in breast and ovarian tumors when compared to their expression across normal tissues. To determine the functional impact of these concomitant amplification and focal deletion events, we first leveraged open-reading-frame (ORF) screen data from 16 cancer cell lines, where we observed that overexpression of BRD4-long and BRD4-short isoforms is toxic for cancer cell growth. We confirmed these results in OVSAHO ovarian cancer cells, where overexpression of both isoforms significantly reduced tumor growth. Next, we mimicked the focal deletions occurring in BRD4 regulatory regions by CRISPR-Cas9 technology, and observed that their depletion functionally ablates tumor cell growth. We finally show that these focal deletions rescue ovarian carcinoma cells from the toxicity effects associated with gene overexpression, suggesting that global BRD4 gene expression levels must be fine tuned to ensure proper cancer cell proliferation. Our study provides experimental evidence for BRD4 deletions constituting the first example of a driver SV alteration reducing toxicity in cancer, therefore expanding the landscape of cancer progression mechanisms.

cancer biology↗

bioRxiv2024

The evolutionarily conserved PhLP3 is essential for sperm development in Drosophila melanogaster

Phosducin-like proteins (PhLP) are thioredoxin domain-containing proteins that are highly-conserved across unicellular and multicellular organisms. PhLP family proteins are hypothesized to function as co-chaperones in the folding of cytoskeletal proteins. Here, we present the initial molecular, biochemical, and functional characterization of CG4511 as Drosophila melanogaster PhLP3. We cloned the gene into a bacterial expression vector and produced enzymatically active recombinant PhLP3, which showed similar kinetics to previously characterized orthologues. A fly strain homozygous for a P-element insertion in the 5 UTR of the PhLP3 gene exhibited significant downregulation of PhLP3 expression. We found these male flies to be sterile. Microscopic analysis revealed altered testes morphology and impairment of spermiogenesis, leading to a lack of mature sperm. Among the most significant observations was the lack of actin cones during sperm maturation. Excision of the P-element insertion in PhLP3 restored male fertility, spermiogenesis, and seminal vesicle size. Given the high level of conservation of PhLP3, our data suggests PhLP3 may be an important regulator of sperm development across species.

biochemistry↗

bioRxiv2023

Double-strand break repair-associated intragenic deletions and tandem duplications suggest the architecture of the repair replication fork

Double-strand break (DSB) repair is associated with a 1000-fold increase in mutations compared to normal replication of the same sequences. In budding yeast, repair of an HO endonuclease-induced DSB at the MAT locus can be repaired by using a homologous, heterochromatic HMR::Kl-URA3 donor harboring a transcriptionally silenced URA3 gene, resulting in a MAT::URA3 (Ura+) repair product where URA3 is expressed. Repair-associated ura3- mutations can be selected by resistance to 5-fluoroorotic acid (FOA). Using this system, we find that a major class of mutations are -1 deletions, almost always in homonucleotide runs, but there are few +1 insertions. In contrast, +1 and -1 insertions in homonucleotide runs are nearly equal among spontaneous mutations. Approximately 10% of repair-associated mutations are interchromosomal template switches (ICTS), even though the K. lactis URA3 sequence embedded in HMR is only 72% identical with S. cerevisiae ura3-52 sequences on a different chromosome. ICTS events begin and end in regions of short microhomology, averaging 7 bp. Long microhomologies are favored, but some ICTS junctions are as short as 2 bp. Both repair-associated intragenic deletions (IDs) and tandem duplications (TDs) are recovered, with junctions sharing short stretches of, on average, 6 bp of microhomology. Intragenic deletions are more than 5 times more frequent than TDs. IDs have a mean length of 60 bp, but, surprisingly there are almost no deletions shorter than 25 bp. In contrast, TDs average only 12 bp. The usage of microhomologies among intragenic deletions is not strongly influenced by the degree of adjacent homeology. Together, these data provide a picture of the structure of the repair replication fork. We suggest that IDs and TDs occur within the migrating D-loop in which DNA polymerase {delta} copies the template, where the 3 end of a partly copied new DNA strand can dissociate and anneal with a single-stranded region of microhomology that lies either in front or behind the 3 end, within the open structure of a migrating D-loop. Our data suggest that [~]100 bp ahead of the polymerase is "open," but that part of the repair replication apparatus remains bound in the 25 bp ahead of the newly copied DNA, preventing annealing. In contrast, the template region behind the polymerase appears to be rapidly reannealed, limiting template switching to a very short region.

genetics↗

bioRxiv2022

Genomics highlight an underestimation of the urban heat island effect on red oak phenology

The phenological timing of leaf-out in temperate forests is a critical transition point each year, altering the global climate system via changes to carbon and hydrologic cycles and land-surface albedo. In turn, climate is impacting phenology by advancing leaf-out an average of 2.8 +/-0.35 days per decade as the planet warms. Thus, understanding the relationship between warming and leaf-out is critical for understanding future global change. Rural-to-urban gradients in temperature, which result in clines over which urban areas are up to 4{degrees}C warmer than their surrounding countryside (dubbed the urban heat island - UHI - effect), may be used as a space-for-time substitution in studies of response to climate change. However, studies have recently highlighted that using the UHI effect over space rather than measurements at the same site through time results in consistently weaker relationships between temperature and leaf-out date in spring (i.e., urban-to-rural gradients underpredict the impact of warming on leaf-out). While such studies suggest many potential environmental explanations, the effect of genetic diversity is often neglected. While sensitive to environmental warming, leaf-out phenology is also highly heritable. Given that rural areas are largely natural sites, they likely have higher intraspecific genetic diversity than urban sites, where plants are selected by land managers for a narrow set of resilience traits. Here we measured the environment, genomic background, and phenological timing of northern red oak (Q. rubra) over several years between an urban and rural site to demonstrate how genetic background explains why the UHI effect leads to an underprediction of plant response to warming. Using a space-for-time substitution, we found that the date of leaf-out at our sites is predicted to advance approximately 20 days over the next 80 years. However, if we further account for the genomic background at the two sites, leaf-out, phenology is predicted to advance 22 days; a 10% difference between the two models. We demonstrate that this stronger relationship is because urban trees are largely a monoculture and, moreover, are most closely related to individuals at the rural site that leaf out latest. We highlight the critical need to consider genetic background, particularly in studies examining highly heritable traits, because both environment and genetics are changing across rural-to-urban gradients.

plant biology↗

bioRxiv2022

A robust and fast two-sample test of equal correlations with an application to differential co-expression

A robust and fast two-sample test for equal Pearson correlation coefficients (PCCs) is important in solving many biological problems, including, e.g., analysis of differential co-expression. However, few existing methods for this test can achieve robustness against deviation from normal distributions, accuracy under small sample sizes, and computational efficiency simultaneously. Here, we propose such a method for testing DIfferential COrrelation using a Saddlepoint Approximation of the Residual bootstrap (DICOSAR). To achieve robustness, accuracy, and efficiency, DICOSAR combines the ideas underlying the pooled residual bootstrap, the signed root of a likelihood ratio statistic, and a multivariate saddlepoint approximation. Through a comprehensive simulation study and a real data analysis of gene co-expression, we demonstrate that DICOSAR is accurate and robust in controlling the type I error rate for detecting differential correlation and provides a faster alternative to the permutation method. We further show that it can also be used for testing differential correlation matrices. These results suggest that DICOSAR provides an analytical approach to facilitate rapid testing for the equality of PCCs in a large-scale analysis.

bioinformatics↗

bioRxiv2021

Force-induced changes of PilY1 drive surface sensing by Pseudomonas aeruginosa

During biofilm formation, the opportunistic pathogen Pseudomonas aeruginosa uses its type IV pili (TFP) to sense a surface, eliciting increased second messenger production and regulating target pathways required to adapt to a surface lifestyle. The mechanisms whereby TFP detect surface contact is still poorly understood, although mechanosensing is often invoked with little data supporting this claim. Using a combination of molecular genetics and single cell analysis, with biophysical, biochemical and genomics techniques we show that force-induced changes mediated by the von Willebrand A (vWA) domain-containing, TFP tip-associated protein PilY1 are required for surface sensing. Atomic force microscopy shows that PilY1 can undergo force-induced, sustained conformational changes akin to those observed for mechanosensitive proteins like titin. We show that mutation of a single cysteine residue in the vWA domain results in modestly lower surface adhesion forces, increased nanospring-like properties, as well as reduced c-di-GMP signaling and biofilm formation. Mutating this cysteine has allowed us to genetically separate TFP function in twitching from surface sensing signaling. The conservation of this Cys residue in all P. aeruginosa PA14 strains, and its absence in the ~720 sequenced strains of P. aeruginosa PAO1, could contribute to explaining the observed differences in surface colonization strategies observed for PA14 versus PAO1. ImportanceMost bacteria live on abiotic and biotic surfaces in surface-attached communities known as biofilms. Surface sensing and increased levels of the second messenger molecule c-di-GMP are crucial to the transition from planktonic to biofilm growth. The mechanism(s) underlying TFP-mediated surface detection that triggers this c-di-GMP signaling cascade are unclear. Here, we provide a key insight into this question: we show that the eukaryotic-like, vWA domain of the TFP tip-associated protein PilY1 responds to mechanical force, which in turn drives production of a key second messenger needed to regulate surface behaviors. Our studies highlight a potential mechanism that could account for differing surface colonization strategies.

microbiology↗

bioRxiv2020

Fine tuning cyclic-di-GMP signaling in Pseudomonas aeruginosa using the type 4 pili alignment complex

To initiate biofilm formation it is critical for bacteria to sense a surface and respond precisely. Type 4 pili (T4P) have been shown to be important in surface sensing, however, mechanism(s) driving downstream changes important for the switch to biofilm growth have not been clearly defined. Here, using macroscopic bulk assays and single cell tracking analyses of Pseudomonas aeruginosa, we uncover a new role of the T4P alignment complex protein, PilO, in modulating the activity of the diguanylate cyclase (DGC) SadC. Two hybrid and bimolecular fluorescence complementation assays show that PilO physically interacts with SadC and that the PilO-SadC interaction inhibits SadCs activity resulting in decreased biofilm formation and increased motility. We show that disrupting the PilO-SadC interaction contributes to greater variation of cyclic-di-GMP levels among cells, thereby increasing cell-to-cell heterogeneity in the levels of this signal. Thus, this work shows that P. aeruginosa uses a component of the T4P scaffold to fine-tune the levels of this nucleotide signal during surface commitment. Finally, given our previous findings linking SadC to the flagellar machinery, we propose that this DGC acts as a bridge to integrate T4P and flagellar-derived input signals during initial surface engagement. Significance StatementT4P of P. aeruginosa are important for surface sensing and regulating intracellular cyclic-di-GMP levels. This work identifies a new role for the T4P alignment complex, previously known for its role in supporting pili biogenesis, in surface-dependent signaling. Furthermore, our findings indicate that P. aeruginosa uses a single DGC, via a complex web of protein-protein interactions, to integrate signaling through the T4P and the flagellar motor to fine-tune cyclic-di-GMP levels. A key implication of this work is that more than just regulating signal levels, cells must modulate the dynamic range of cyclic-di-GMP to precisely control the transition to a biofilm lifestyle.

microbiology↗