Biology subjects

Feng, W.

Publications and source records attributed to Feng, W..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Structural insights into ligand recognition and activation of the melanocortin-4 receptor

Melanocortin-4 receptor (MC4R) plays a central role in the regulation of energy homeostasis. Its high sequence similarity to other MC receptor family members, low agonist selectivity and the lack of structural information concerning receptor activation have hampered the development of MC4R-seletive therapeutics to treat obesity. Here, we report four high-resolution structures of full-length MC4R in complex with the heterotrimeric Gs protein stimulated by the endogenous peptide -MSH, FDA-approved drugs afamelanotide (Scenesse) and bremelanotide (Vyleesi), and selective small-molecule ligand THIQ, respectively. Together with pharmacological studies, our results reveal the conserved binding mode of peptidic agonists, the distinctive molecular details of small-molecule agonist recognition underlying receptor subtype selectivity, and distinct activation mechanism for MC4R, thereby offering new insights into G protein coupling. Our work may facilitate the discovery of selective therapeutic agents targeting MC4R.

molecular biology↗

bioRxiv2021

Control of spinal motor neuron terminal differentiation through sustained Hoxc8 gene activity

Spinal motor neurons (MNs) constitute cellular substrates for several movement disorders. Although their early development has received much attention, how spinal MNs become and remain terminally differentiated is poorly understood. Here, we determined the transcriptome of mouse brachial MNs at embryonic and postnatal stages. We found that genes encoding homeodomain (HOX, LIM) transcription factors (TFs), previously implicated in early MN development, continue to be expressed postnatally, suggesting later functions. To test this, we inactivated Hoxc8 at successive stages of MN development. We found that Hoxc8 is not only required to establish but also maintain expression of several MN terminal differentiation markers. Furthermore, we uncovered novel TFs with continuous MN expression, a Hoxc8 dependency for maintained expression of Iroquois (Irx) homeodomain TFs, and a new role for Irx2 in MN development. Our findings dovetail recent observations in C. elegans MNs, pointing toward an evolutionarily conserved role for Hox in neuronal terminal differentiation.

developmental biology↗

bioRxiv2021

Structures of the human cholecystokinin receptors in complex with agonists and antagonists

Cholecystokinin receptors, CCKAR and CCKBR, are important neuro-intestinal peptide hormone receptors and play a vital role in food intake and appetite regulation. Here we report three crystal structures of the human CCKAR in complex with different ligands, including one peptide agonist and two small-molecule antagonists, as well as two cryo-electron microscopy structures of CCKBR-gastrin in complex with Gi2 and Gq, respectively. These structures reveal the recognition pattern of different ligand types and the molecular basis of peptide selectivity in the cholecystokinin receptor family. By comparing receptor structures in different conformational states, a stepwise activation process of cholecystokinin receptors is proposed. Combined with pharmacological data, our results provide atomic details for differential ligand recognition and receptor activation mechanisms. These insights will facilitate the discovery of potential therapeutics targeting cholecystokinin receptors.

biophysics↗

bioRxiv2021

FMRP bridges R-loops and DHX9 through direct interactions

Mutations in, or deficiency of, FMRP is responsible for the Fragile X syndrome (FXS), the most common cause for inherited intellectual disability. FMRP is a nucleocytoplasmic protein, primarily characterized as a translation repressor with poorly understood nuclear function(s). We recently uncovered a genome protective role of FMRP. We reported that FXS patient-derived cells lacking FMRP sustain higher level of DNA double-strand breaks than normal cells, a phenotype further exacerbated by DNA replication stress. The stress-induced DSBs occur at sequences prone to form R-loops, which are co-transcriptional RNA:DNA hybrids that have been associated with genome instability. Concordantly, we showed that FXS cells accumulate R-loops under replication stress. Moreover, expression of FMRP and not a mutant deficient in binding nucleic acids and known to cause FXS, FMRPI304N, reduced R-loop-associated DSBs. These observations demonstrated that FMRP promotes genome integrity by preventing R-loop accumulation and chromosome breakage. Here, we explore the mechanism through which FMRP prevents R-loop accumulation in an isogenically controlled CRISPR KO of FMR1 (gene encoding for FMRP) in HEK293T cells. We demonstrate for the first time that FMRP directly binds R-loops. We show that FMRP interacts with DHX9, an RNA helicase that unwinds both double strand RNA and RNA:DNA hybrids and regulates R-loop formation through modulating these activities. This interaction is reduced with FMRPI304N, suggesting that FMRP regulation of R-loop is mediated through DHX9. Interestingly, we show that FMRP inhibits DHX9 helicase activity on RNA:DNA hybrids. Moreover, DHX9 binds chromatin containing R-loops more efficiently in the absence of a functional FMRP. These results suggest an antagonistic relationship between FMRP and DHX9 at the chromatin, where FMRP prevents R-loop formation by suppressing DHX9. Our study sheds new light on our understanding of the genome functions of FMRP.

biochemistry↗

bioRxiv2021

LIN37-DREAM Prevents DNA End Resection and Homologous Recombination at DNA Double Strand Breaks in Quiescent Cells

DNA double strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to expression of HR proteins, including BRCA1, BRCA2, PALB2 and RAD51, and DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.

cell biology↗

bioRxiv2021

Changes and composition of microbial community during aerobic composting of household food waste

In order to explore the effect of added bacteria on microbial community and determine the dominant bacteria in the aerobic composting process of household food waste (HFW), two groups of HFW composting experiments were conducted for 30 days. The final degradation rates of the two groups were 69.95% (group A, natural composting) and 73.52% (Group C, composting with added bacteria), respectively. 16S rRNA high-throughput sequencing was used to analyze the changes of microbial community in the composting process. As the result displays, at the classification of phylum level, the relatively abundant bacteria of two groups were Firmicutes, Proteobacteria and Cyanobacteria. At the classification of genus level, Pediococcus is the dominant bacteria of group A, which performed better in maintaining the microbial community stable in the later stage of composting, while Weissella accounted for a relatively large proportion of group C and behaved well in promoting the final degradation rate of composting. The proportion of Ochrobactrum in the early stage of group C is considerable and it is closely related to the removal of odour during composting. The relative abundance of added bacteria was always in a rather low level, suggested that the way they affect the composting process is to change the proportion of dominant bacteria in different stage of composting. This experiment provides an important reference for improving the microbial degradation efficiency of HFW. IMPORTANCEIn recent years, food waste has gradually become a global problem, the annual waste of food is as high as 1.3 billion tons. FW, especially HFW, as a high content of organic matter waste, has a pretty good recycling value. So how to deal with and recycle it efficiently, quickly and conveniently becomes more and more important. Among many treatment and recovery methods, microbial treatment (including anaerobic digestion and aerobic composting) has gradually become a research hotspot due to its advantages of low pollution and low energy consumption, and microorganisms play a crucial role in these process. In this study we use 16S rRNA high-throughput sequencing method to explore aerobic composting of HFW. The purpose of this study is to find out the dominant bacteria which can improve the degradation efficiency, remove the odor and prolong the treatment cycle, and then provide further theoretical reference for future HFW related research.

microbiology↗

bioRxiv2021

Exceptional origin activation revealed by comparative analysis in two laboratory yeast strains

We performed a comparative analysis of replication origin activation by genome-wide single-stranded DNA mapping in two common laboratory strains of Saccharomyces cerevisiae challenged by hydroxyurea (HU), an inhibitor of the ribonucleotide reductase. By doing so we gained understanding of the impact on origin activation by three factors: replication checkpoint control, DNA sequence polymorphisms, and relative positioning of origin and transcription unit. Our analysis recapitulated the previous finding that the majority of origins are subject to checkpoint control by the Rad53 kinase when cells were treated with HU. In addition, origins either subject to Rad53 checkpoint control or impervious to it are largely concordant between the two strains. However, these two strains also produced different dynamics of origin activation. First, the W303-RAD53 cells showed a significant reduction of fork progression than A364a-RAD53 cells. This phenotype was accompanied by an elevated level of Rad53 phosphorylation in W303-RAD53 cells. Second, W303-rad53K227A checkpoint-deficient cells activated a greater number of origins accompanied by global reduction of ssDNA across all origins compared to A364a-rad53K227A cells; and this is correlated with lower expression level of the mutant protein in W303 than in A364a. We also show that sequence polymorphism in the consensus motifs of the replication origins plays a minor role in determining origin usage. Remarkably, eight strain-specific origins lack the canonical 11-bp consensus motif for autonomously replicating sequences in either strain background. Finally, we identified a new class of origins that are only active in checkpoint-proficient cells, which we named "Rad53-dependent origins". The only discernible feature of these origins is that they tend to overlap with an open reading frame, suggesting previously unexplored connection between transcription and origin activation. Our study presents a comprehensive list of origin usage in two diverse yeast genetic backgrounds, fine-tunes the different categories of origins with respect to checkpoint control, and provokes further exploration of the interplay between origin activation and transcription. Author SummaryComparative analysis of origins of replication in two laboratory yeast strains reveals new insights into origin activation, regulation and dependency on the Rad53 checkpoint kinase.

genomics↗

bioRxiv2021

A water-reward task assay for evaluating mouse mutualistic cooperative behavior

Social cooperation is fundamentally important for group animals but rarely studied with mice because of their natural aggressiveness. In the present work, we induced pairs of mice to develop a mutualistic cooperative behavior in a non-divided chamber. Each mouse was first trained to learn to use a water dispenser by occupying a particular zone served as a switch to the dispenser. Two trained mice were then put into a chamber containing two separate zones jointly controlling two dispensers. We recorded the latency before each co-drinking, the number and cumulated time of co-drinking each day during the test. These parameters served as quantitative measurements of cooperative behavior in mice. The whole procedure includes preparation, training and testing phases, which take 15 days in total. This assay provides detailed procedures and analytical methods for investigators to characterize and quantify the mutualistic cooperative behavior. The use of mice as subjects allows convenient coupling to other behavior assays and is amiable to genetic manipulations for mechanistic study.

animal behavior and cognition↗

bioRxiv2020

Computational profiling of hiPSC-derived heart organoids reveals chamber defects associated with Ebstein's anomaly

Heart organoids have the potential to generate primary heart-like anatomical structures and hold great promise as in vitro models for cardiac disease. However, their properties have not yet been carefully studied, which hinders a wider spread application. Here we report the development of differentiation systems for ventricular and atrial heart organoids, enabling the study of heart disease with chamber defects. We show that our systems generate organoids comprising of major cardiac cell types, and we used single cell RNA sequencing together with sample multiplexing to characterize the cells we generate. To that end, we also developed a machine learning label transfer approach lever-aging cell type, chamber, and laterality annotations available for primary human fetal heart cells. We then used this model to analyze organoid cells from an isogeneic line carrying an Ebsteins anomaly associated genetic variant, and we successfully recapitulated the diseases atrialized ventricular defects. In summary, we have established a workflow integrating heart organoids and computational analysis to model heart development in normal and disease states.

developmental biology↗

bioRxiv2020

Effects of Ca2+ on encystment and growth in Scrippsiella trochoidea

Cysts serve as a seed source for the initiation and recurrence of a harmful algal bloom (HAB) caused by dinoflagellates. And the influence of calcium on cyst formation has been relatively understudied. In the present study, we investigated the effects of calcium (Ca2+) on the growth and encystment of Scrippsiella trochoidea. We incubated S. trochoidea in modified f/2 media in flasks which were divided into five groups and treated with different Ca2+ concentrations (0, 0.2, 0.4, 0.6, and 0.8 g{middle dot}L-1). We revealed that cell density increased with increasing Ca2+ concentrations; however, cell density was reduced when Ca2+ concentrations exceeded 0.2 g{middle dot}mL-1. Additionally, the number of cysts and the cyst formation rate similarly increased as Ca2+ concentrations increased, but these were reduced when Ca2+ concentrations exceeded 0.4 g{middle dot}mL-1. Lastly, S. trochoidea absorbed Ca2+ from the water when cysts were formed and under high Ca2+ concentrations, more calcareous thorn cysts formed.

ecology↗

bioRxiv2020

Pulmonary B cells ameliorate Alzheimer's disease-like neuropathology

Increasing evidence shows that the peripheral immune system is involved in the pathogenesis of Alzheimers disease (AD). Here, we report that pulmonary B cells mitigate beta-Amyloid (A{beta}) pathology in 5xFAD mice. The proportion of B cells rather than T cells increases in brain, meningeal and lung tissues in 3-month-old 5xFAD mice. Deletion of B cells aggravates A{beta} load and memory deficits of 5xFAD mice. Mechanimsly, pulmonary B cells can migrate to the brain parenchyma and produce interleukin-35 that inhibits neuronal {beta}-site APP-cleaving enzyme 1 expression, subsequently reducing the production of A{beta}. In turn, proliferation of pulmonary B cells is associated with activation of toll-like receptor/nuclear factor kappa-B pathway by elevated A{beta} that is drained from the brain parenchyma to the lungs via meningeal lymphatics. Furthermore, promoting pulmonary B cell proliferation via overexpression of B-cell-activating factor ameliorates brain A{beta} load and improves cognitive functions of 10-month-old 5xFAD mice. Together, these results highlight the lungs as both immune targets and effector organs in A{beta} pathogenesis. Pulmonary B cells might be a potential target against AD.

neuroscience↗

bioRxiv2020

Non-lesioned subcortical brain volumes are associated with post-stroke sensorimotor behavior across 28 cohorts worldwide: An ENIGMA Stroke Recovery study

Background and PurposeUp to two-thirds of stroke survivors experience persistent sensorimotor impairments. Recovery relies on the integrity of spared brain areas to compensate for damaged tissue. Subcortical regions play critical roles in the control and regulation of sensorimotor circuits. The goal of this work is to identify associations between volumes of spared subcortical nuclei and sensorimotor behavior at different timepoints after stroke. MethodsWe pooled high-resolution T1-weighted MRI brain scans and behavioral data in 828 individuals with unilateral stroke from 28 cohorts worldwide. Cross-sectional analyses using linear mixed-effects models related post-stroke sensorimotor behavior to non-lesioned subcortical volumes (Bonferroni-corrected, p<0.004). We tested subacute ([≤]90 days) and chronic ([≥]180 days) stroke subgroups separately, with exploratory analyses in early stroke ([≤]21 days) and across all time. Sub-analyses in chronic stroke were also performed based on class of sensorimotor deficits (impairment, activity limitations) and side of lesioned hemisphere. ResultsWorse sensorimotor behavior was associated with a smaller ipsilesional thalamic volume in both early (n=179; d=0.68) and subacute (n=274, d=0.46) stroke. In chronic stroke (n=404), worse sensorimotor behavior was associated with smaller ipsilesional putamen (d=0.52) and nucleus accumbens (d=0.39) volumes, and a larger ipsilesional lateral ventricle (d=-0.42). Worse chronic sensorimotor impairment specifically (measured by the Fugl-Meyer Assessment; n=256) was associated with smaller ipsilesional putamen (d=0.72) and larger lateral ventricle (d=-0.41) volumes, while several measures of activity limitations (n=116) showed no significant relationships. In the full cohort across all time (n=828), sensorimotor behavior was associated with the volumes of the ipsilesional nucleus accumbens (d=0.23), putamen (d=0.33), thalamus (d=0.33), and lateral ventricle (d=-0.23). ConclusionsWe demonstrate significant relationships between post-stroke sensorimotor behavior and reduced volumes of subcortical gray matter structures that were spared by stroke, which differ by time and class of sensorimotor measure. These findings may provide additional targets for improving post-stroke sensorimotor outcomes.

neuroscience↗

bioRxiv2020

Targeted DNA Damage Repair CRISPR/Cas9 Knockout Screen Identifies Novel Classification of Poly-ADP Ribose Polymerase Inhibitors Based on Key Base Excision Repair Proteins

DNA repair deficiencies have become an increasingly promising target for novel therapeutics within the realm of clinical oncology. Recently, several inhibitors of Poly(ADP-ribose) Polymerases (PARPs) have received approval for the treatment of cancers primarily with deleterious mutations in the homologous recombination (HR) proteins, BRCA1 and BRCA2. Despite numerous clinical trials which have been completed or are currently ongoing, the mechanism of action by which PARP inhibitors selectively kill tumor cells is poorly understood. While many believe "trapping" of PARP proteins to DNA at sites of damage is the most important determinant driving cytotoxicity by these drugs, clinically effective inhibitors exist with a diverse range of PARP-trapping qualities. These findings suggest that characterization of inhibitors as strong versus weak trappers does not properly capture the intra-class characteristics of these drugs. Here, we use a novel, targeted DNA damage response (DDR) CRISPR/Cas9 screening library to reveal heterogenous genetic dependencies on the base excision repair (BER) pathway for PARP inhibitors, which is not correlated with PARP trapping ability or catalytic inhibition of PARP. These findings demonstrate that inhibition of PARylation and induction of PARP trapping are not the only factors contributing to distinct biological activity for different PARP inhibitors, and they provide insight into the optimal choice of PARP inhibitors for use in the setting of specific DDR defects. AUTHOR SUMMARYTargeted cancer therapies rely on our general understanding of which genetic mutations are involved in both sensitivity and resistance to such anticancer agents. In this study, we describe the use of functional genetic screening to evaluate the role of various DNA repair proteins in response to inhibitors of PARP, a quintessential example of targeted therapy. While PARP inhibitors are best known for their utility in cancers with homologous recombination defects, we show that some inhibitors within this class may have additional functionality in cancers with deficient base excision repair. These findings highlight not only the importance of PARP inhibitor selection in the appropriate context, but also the mechanistic differences that exist within this class of inhibitors. It is our hope that our findings will inspire future work evaluating the use of specific PARP inhibitor selection in designing clinical trials to further expand the use of PARP inhibitors beyond tumors with homologous recombination deficiencies.

cancer biology↗

bioRxiv2020

SETD2 deficiency impairs β-catenin destruction complex to facilitate renal cell carcinoma formation

Clear cell renal cell carcinoma (ccRCC) is a largely incurable disease that is highly relevant to epigenetic regulation including histone modification and DNA methylation. SET domain-containing 2 (SETD2) is a predominant histone methyltransferase catalyzing the trimethylation of histone H3 Lysine 36 (H3K36me3) and its mutations are highly relevant to clear cell renal cell carcinoma (ccRCC). However, its physiology role in ccRCC remains largely unexplored. Here we report that Setd2 deletion impairs the {beta}-catenin destruction complex to facilitate ccRCC formation in a c-MYC-generated polycystic kidney disease (PKD) model, which can be relieved by an inhibitor of {beta}-catenin-responsive transcription. Clinically, SETD2 loss is widely observed in ccRCC samples, and negatively correlated with expression of some members of {beta}-catenin destruction complex, but positively correlated with the activation of Wnt/{beta}-catenin signaling. Our findings thus highlight a previously unrecognized role of SETD2-mediated H3K36me3 modification in regulation of Wnt/{beta}-catenin pathway in ccRCC. SummaryOur findings for the first time reveal a previously unrecognized role of the SETD2-mediated H3K36me3 modification in regulation of the Wnt/{beta}-catenin pathway in ccRCC and shed light on the molecular mechanisms underlying the formation of renal cell carcinoma with epigenetic disorders.

cancer biology↗

bioRxiv2020

The Histone Methyltransferase SETD2 Modulates Oxidative Stress to Attenuate Colonic Inflammation and Tumorigenesis in Mice

BACKGROUND & AIMSInflammatory bowel disease (IBD) is a complex and relapsing inflammatory disease, and patients with IBD exhibit a higher risk of developing colorectal cancer (CRC). Epithelial barrier disruption is one of the major causes of IBD in which epigenetic modulation is pivotal. However, the epigenetic mechanisms underlying the epithelial barrier integrity regulation remain largely unexplored. Here, we investigated how SETD2, an epigenetic modifier, maintains intestinal epithelial homeostasis and attenuates colonic in[fl]ammation and tumorigenesis. METHODSGEO public database and IBD tissues were used to investigate the clinical relevance of SETD2 in IBD. To define a role of SETD2 in the colitis, we generated mice with epithelium-specific deletion of Setd2 (Setd2Vil-KO mice). Acute colitis was induced by 2% dextran sodium sulfate (DSS), and colitis-associated CRC was induced by injecting azoxymethane (AOM), followed by three cycles of 2% DSS treatments. Colon tissues were collected from mice and analyzed by histology, immunohistochemistry and immunoblots. Organoids were generated from Setd2Vil-KO and control mice, and were stained with 7-AAD to detect apoptosis. A fluorescent probe, 2',7'-dichlorodihydro[fl]uorescein diacetate (H2DCFDA), was used to detect the levels of ROS in intestinal epithelial cells (IECs) isolated from the two types of mice. RNA-seq and H3K36me3 ChIP-seq analyses were performed to identify the mis-regulated genes modulated by SETD2. Results were validated in functional rescue experiments by N-acetyl-l-cysteine (NAC) treatment and transgenes expression in IECs. RESULTSSETD2 expression became decreased in IBD patients and DSS-treated colitis mice. Setd2Vil-KO mice displayed abnormal loss of mucus-producing goblet cells and antimicrobial peptide (AMP)-producing Paneth cells, and exhibited pre-mature intestinal inflammation development. Consistent with the reduced SETD2 expression in IBD patients, Setd2Vil-KO mice showed increased susceptibility to DSS-induced colitis, accompanied by more severe epithelial barrier disruption and markedly increased intestinal permeability that subsequently facilitated in[fl]ammation-associated CRC. Mechanistically, deletion of Setd2 resulted in excess reactive oxygen species (ROS), which led to cellular apoptosis and defects in barrier integrity. NAC treatment in Setd2Vil-KO mice rescued epithelial barrier injury and apoptosis. Importantly, Setd2 depletion led to excess ROS by directly down-regulating antioxidant genes that inhibit ROS reaction. Moreover, overexpression of antioxidant PRDX6 in Setd2Vil-KO IECs largely alleviated the overproductions of ROS and improved the cellular survival. CONCLUSIONSDeficiency of Setd2 specifically in the intestine aggravates epithelial barrier disruption and inflammatory response in colitis via a mechanism dependent on oxidative stress. Thus, our results highlight an epigenetic mechanism by which Setd2 modulates oxidative stress to regulate intestinal epithelial homeostasis. SETD2 might therefore be a pivotal regulator that maintains the homeostasis of the intestinal mucosal barrier.

cell biology↗

bioRxiv2020

Single-cell screening of SARS-CoV-2 target cells in pets, livestock, poultry and wildlife

A few animals have been suspected to be intermediate hosts of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, a large-scale single-cell screening of SARS-CoV-2 target cells on a wide variety of animals is missing. Here, we constructed the single-cell atlas for 11 representative species in pets, livestock, poultry, and wildlife. Notably, the proportion of SARS-CoV-2 target cells in cat was found considerably higher than other species we investigated and SARS-CoV-2 target cells were detected in multiple cell types of domestic pig, implying the necessity to carefully evaluate the risk of cats during the current COVID-19 pandemic and keep pigs under surveillance for the possibility of becoming intermediate hosts in future coronavirus outbreak. Furthermore, we screened the expression patterns of receptors for 144 viruses, resulting in a comprehensive atlas of virus target cells. Taken together, our work provides a novel and fundamental strategy to screen virus target cells and susceptible species, based on single-cell transcriptomes we generated for domesticated animals and wildlife, which could function as a valuable resource for controlling current pandemics and serve as an early warning system for coping with future infectious disease threats.

cell biology↗

bioRxiv2020

Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

TP53 mutations in cancer are associated with poor patient outcomes and resistance to DNA damaging therapies1-3. However, the mechanisms underlying treatment resistance in p53-deficient cells remain poorly characterized. Here, we show that p53-deficient cells exhibit hyperactive repair of therapy-induced DNA double strand breaks (DSBs), which is suppressed by inhibition of DNA-dependent protein kinase (DNA-PK). Single-cell analyses of DSB repair kinetics and cell cycle state transitions reveal an essential role for DNA-PK in suppressing S phase DNA damage and mitotic catastrophe in p53-deficient cells. Yet, a subset of p53-deficient cells exhibit intrinsic resistance to therapeutic DSBs due to a repair pathway that is not sensitive to DNA-PK inhibition. We show that p53 deficiency induces overexpression of DNA Polymerase Theta (Pol {theta}), which mediates an alternative end-joining repair pathway that becomes hyperactivated by DNA-PK inhibition4. Combined inhibition of DNA-PK and Pol {theta} restores therapeutic DNA damage sensitivity in p53-deficient cells. Thus, our study identifies two targetable DSB end joining pathways that can be suppressed as a strategy to overcome resistance to DNA-damaging therapies in p53-deficient cancers.

cell biology↗

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↗