PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents.Spanish Ministry of Science, Innovation and Universities (grants BFU2016-80402R and BFU2019-106707-RB to JM and PGC2018.093576-B-C21 to LB, co-sponsored by EU ERDF funds), the Swiss National Science Foundation (grant 31003A_169959 to ML), and an ERC Consolidator Grant (617102 to ML

Characterizing the tumor microenvironment in rare renal cancer histological types

The tumor microenvironment (TME), including immune cells, cancer-associated fibroblasts, endothelial cells, adjacent normal cells, and others, plays a crucial role in influencing tumor behavior and progression. Here, we characterized the TME in 83 primary renal tumors and matched metastatic or recurrence tissue samples (n = 15) from papillary renal cell carcinoma (pRCC) types 1 (n = 20) and 2 (n = 49), collecting duct carcinomas (CDC; n = 14), and high-grade urothelial carcinomas (HGUC; n = 5). We investigated 10 different markers of immune infiltration, vasculature, cell proliferation, and epithelial-to-mesenchymal transition by using machine learning image analysis in conjunction with immunohistochemistry. Marker expression was compared by Mann-Whitney and Kruskal-Wallis tests and correlations across markers using Spearman's rank correlation coefficient. Multivariable Poisson regression analysis was used to compare marker expression between histological types, while accounting for variation in tissue size. Several immune markers showed different rates of expression across histological types of renal carcinoma. Using pRCC1 as reference, the incidence rate ratio (IRR) of CD3+ T cells (IRR [95% confidence interval, CI] = 2.48 [1.53-4.01]) and CD20+ B cells (IRR [95% CI] = 4.38 [1.22-5.58]) was statistically significantly higher in CDC. In contrast, CD68+ macrophages predominated in pRCC1 (IRR [95% CI] = 2.35 [1.42-3.9]). Spatial analysis revealed CD3+ T-cell and CD20+ B-cell expressions in CDC to be higher at the proximal (p < 0.0001) and distal (p < 0.0001) tumor periphery than within the central tumor core. In contrast, expression of CD68+ macrophages in pRCC2 was higher in the tumor center compared to the proximal (p = 0.0451) tumor periphery and pRCC1 showed a distance-dependent reduction, from the central tumor, in CD68+ macrophages with the lowest expression of CD68 marker at the distal tumor periphery (p = 0.004). This study provides novel insights into the TME of rare kidney cancer types, which are often understudied. Our findings of differences in marker expression and localization by histological subtype could have implications for tumor progression and response to immunotherapies or other targeted therapies

Monitoring the Replication and Repair of Psoralen Induced Interstrand Crosslinks on Human Genomic DNA

Interstrand crosslinks (ICL) represent highly cytotoxic lesions that covalently link two strand of DNA in a duplex, thus posing a threat to replication and transcription, two fundamental processes for life. ICL inducing agents are among the most widely used chemotherapeutics, but unfortunately cancer cells are able to repair ICL using Fanconi Anemia (FA) and Homologous Recombination (HR) proteins, thereby evading or limiting cell death. Based on biochemical work with plasmid DNA and cell-free extracts, ICL were long considered an absolute roadblock to the replication machinery, but a recent study in human cells has shown that the replication machinery can in fact efficiently traverse ICLs. We synthesized a chemically modified psoralen (DIG-TMP) that can be recognized by specific antibodies and exploited single-molecule approaches – i.e. mainly DNA fiber assays and visualization of replication intermediates by Electron Microscopy (EM) - to monitor directly human genome replication across ICLs (local replication), as well as elsewhere in the genome (global replication). Performing this parallel analysis in different genetic backgrounds (i.e. upon inactivation of the crucial FA factors FANCM, FANCD2 and RAD51, as well as the fork remodeling factor ZRANB3), we found that (i) the fork-remodeling factor RAD51 is immediately recruited to stressed replication forks before any detectable double stranded break formation, (ii) replication fork reversal is very frequent upon DIG-TMP treatment, assists ICL traverse and prevents premature SLX4-mediated chromosomal breakage, (iii) global fork slowdown upon ICL induction is associated with fork reversal and depends upon ATR activity. I also developed Immuno Electron Microscopy (iEM) to directly visualize ICLs on individual replication intermediates: albeit technically challenging and currently not applicable for large scale analysis, this approach showed potential to reveal architectural changes of replication forks facing ICLs and to assess a direct functional link between fork remodeling and ICL bypass or repair. This study provides important mechanistic insight on ICL replication and repair and contributes to clarify resistance mechanisms upon cancer chemotherapeutic regimens with ICL-inducing agents

Efficacy of Zapotin as a Therapeutic Drug Against Non Small Cell Lung Cancer

Lung cancer has been the leading cause of cancer related deaths around the world. The most common type of lung cancer, nearly 85% is Non Small Cell lung Cancer (NSCLC). This study provides an evidence of efficacy of Zapotin (5,6,2',6'-tetramethoxyflavone) against NSCLC cell line. Zapotin was found to be effective against A549 cells that express wild type p53. Since Zapotin increased the protein expression of p53 in A549 cells, its mode of action is expected through p53. Target proteins of p53, which are involved in the cell cycle arrest and apoptosis were found to be up regulated upon treatment of A549 cells with Zapotin. Effects of Zapotin were tested on the proteins involved in induction and stabilization of p53 viz. ATM, p300, Mdm2, Mdm4 and Snai1. A549 cells possess oncogeneic K-ras, which suppresses the p53 activity through a Snai1 mediated pathway of p53 degradation. Further translational inhibition studies provided an evidence of p53 stabilization rather than induction of p53 by Zapotin. Our results here indicate restoration of p53 function in NSCLC cells treated with Zapotin through the inactivation of Snai1 in K-Ras mutated cells with wild type p53.M.S. in Biology, May 201

ATR-Mediated Global Fork Slowing and Reversal Assist Fork Traverse and Prevent Chromosomal Breakage at DNA Interstrand Cross-Links

Summary: Interstrand cross-links (ICLs) are toxic DNA lesions interfering with DNA metabolism that are induced by widely used anticancer drugs. They have long been considered absolute roadblocks for replication forks, implicating complex DNA repair processes at stalled or converging replication forks. Recent evidence challenged this view, proposing that single forks traverse ICLs by yet elusive mechanisms. Combining ICL immunolabeling and single-molecule approaches in human cells, we now show that ICL induction leads to global replication fork slowing, involving forks not directly challenged by ICLs. Active fork slowing is linked to rapid recruitment of RAD51 to replicating chromatin and to RAD51/ZRANB3-mediated fork reversal. This global modulation of fork speed and architecture requires ATR activation, promotes single-fork ICL traverse—here, directly visualized by electron microscopy—and prevents chromosomal breakage by untimely ICL processing. We propose that global fork slowing by remodeling provides more time for template repair and promotes bypass of residual lesions, limiting fork-associated processing. : Replication-coupled repair of DNA interstrand cross-links (ICLs) promotes resistance to chemotherapeutic treatments. Visualizing individual lesions and replication intermediates, Mutreja et al. report that forks slow down and reverse both at ICLs and away from lesions. This ATR-mediated response assists lesion bypass during replication and limits chromosomal breakage by fork-associated processing. Keywords: DNA replication, DNA replication stress response, replication fork reversal, fork traverse, DNA interstrand crosslinks, ATR checkpoint, global fork slowing, electron microscopy, ICL immunolabelin

Host, reproductive, and lifestyle factors in relation to quantitative histologic metrics of the normal breast

Abstract Background Emerging data indicate that variations in quantitative epithelial and stromal tissue composition and their relative abundance in benign breast biopsies independently impact risk of future invasive breast cancer. To gain further insights into breast cancer etiopathogenesis, we investigated associations between epidemiological factors and quantitative tissue composition metrics of the normal breast. Methods The study participants were 4108 healthy women ages 18–75 years who voluntarily donated breast tissue to the US-based Susan G. Komen Tissue Bank (KTB; 2008–2019). Using high-accuracy machine learning algorithms, we quantified the percentage of epithelial, stromal, adipose, and fibroglandular tissue, as well as the proportion of fibroglandular tissue that is epithelium relative to stroma (i.e., epithelium-to-stroma proportion, ESP) on digitized hematoxylin and eosin (H&E)-stained normal breast biopsy specimens. Data on epidemiological factors were obtained from participants using a detailed questionnaire administered at the time of tissue donation. Associations between epidemiological factors and square root transformed tissue metrics were investigated using multivariable linear regression models. Results With increasing age, the amount of stromal, epithelial, and fibroglandular tissue declined and adipose tissue increased, while that of ESP demonstrated a bimodal pattern. Several epidemiological factors were associated with individual tissue composition metrics, impacting ESP as a result. Compared with premenopausal women, postmenopausal women had lower ESP [β (95% Confidence Interval (CI)) = −0.28 (− 0.43, − 0.13); P < 0.001] with ESP peaks at 30–40 years and 60–70 years among pre- and postmenopausal women, respectively. Pregnancy [β (95%CI) vs nulligravid = 0.19 (0.08, 0.30); P < 0.001] and increasing number of live births (P -trend < 0.001) were positively associated with ESP, while breastfeeding was inversely associated with ESP [β (95%CI) vs no breastfeeding = −0.15 (− 0.29, − 0.01); P = 0.036]. A positive family history of breast cancer (FHBC) [β (95%CI) vs no FHBC = 0.14 (0.02–0.26); P = 0.02], being overweight or obese [β (95%CI) vs normal weight = 0.18 (0.06–0.30); P = 0.004 and 0.32 (0.21–0.44); P < 0.001, respectively], and Black race [β (95%CI) vs White = 0.12 (− 0.005, 0.25); P = 0.06] were positively associated with ESP. Conclusion Our findings revealed that cumulative exposure to etiological factors over the lifespan impacts normal breast tissue composition metrics, individually or jointly, to alter their dynamic equilibrium, with potential implications for breast cancer susceptibility and tumor etiologic heterogeneity

PrimPol-mediated repriming facilitates replication traverse of DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) induced by endogenous aldehydes or chemotherapeutic agents interfere with essential processes such as replication and transcription. ICL recognition and repair by the Fanconi Anemia pathway require the formation of an X-shaped DNA structure that may arise from convergence of two replication forks at the crosslink or traversing of the lesion by a single replication fork. Here, we report that ICL traverse strictly requires DNA repriming events downstream of the lesion, which are carried out by PrimPol, the second primase-polymerase identified in mammalian cells after Polα/Primase. The recruitment of PrimPol to the vicinity of ICLs depends on its interaction with RPA, but not on FANCM translocase or the BLM/TOP3A/RMI1-2 (BTR) complex that also participate in ICL traverse. Genetic ablation of PRIMPOL makes cells more dependent on the fork convergence mechanism to initiate ICL repair, and PRIMPOL KO cells and mice display hypersensitivity to ICL-inducing drugs. These results open the possibility of targeting PrimPol activity to enhance the efficacy of chemotherapy based on DNA crosslinking agents

The MMS22L-TONSL heterodimer directly promotes RAD51-dependent recombination upon replication stress

Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo

"I got mad about that book" : A study of Vietnamese children's views on reading

The aim of this Bachelor thesis and Minor Field Study is, from the perspective of the Vietnamese children at The General Science Library in Ho Chi Minh City, to obtain a deeper understanding of the ways in which the reading environment of The Children's Room supports children's interest in reading and their reading experience. It is assumed that reading is a dynamic and social activity made possible by internal and external conditions. In this context limited freedom of expression is one of the external conditions that is taken into consideration. In order to obtain understanding of Vietnamese children's experiences of reading, I used Aidan Chambers' model of The Reading Circle as a theoretical framework. The methods used in this study were semi-structured interviews with Vietnamese young people at the library, four girls and five boys, aged between 10 and 15 years. The children in this study describe two types of reading experiences: 1) reading that gives feelings of excitement and joy, and makes them want to reread a book, talk about it with others, think of it, remember and analyse it. And 2) reading they describe as developing, either spiritually or intellectually, a form of reading for improvement. Four reading environments are identified that both enable and obstruct reading experiences, their homes, school, library and the bookstore. Primarily, The Children's Room enables reading