Biology subjects

Taglialatela, A.

Publications and source records attributed to Taglialatela, A..

6 recordsLinked to original sources

bioRxiv2026

Mapping the genetic landscape of the DNA damage response with Cas12a-based combinatorial knockout screens

The DNA damage response (DDR) is a complex network of cellular pathways that ensures the faithful maintenance of our genomes upon a wide array of genomic insults. To elucidate the functional architecture of this network, we conducted unbiased genetic interaction screens using the Cas12a genome editor to disrupt 233 DDR genes frequently mutated in cancer and other genetic diseases, either individually or in pairwise combinations. This approach enabled us to assess the phenotypic effects induced by the disruption of >27,000 DDR gene pair combinations under unperturbed cell growth conditions. From this analysis, we identified over 750 high-confidence positive (buffering) or negative (synthetic lethal/sick) gene-gene interactions, along with multiple connections between previously unlinked DDR pathways and modules, allowing us to define novel aspects of the cellular response to spontaneous, DNA replication-associated DNA damage. Among the identified genetic interactions, we uncovered profound synthetic lethal interactions between genes encoding 1) the translesion polymerase REV1-Pol {zeta} complex and the MCM8-MCM9-HROB DNA helicase complex; 2) Fanconi Anemia (FA) proteins and the mitotic DNA repair factors GEN1, CIP2A, and RHINO; and 3) the DNA translocase SMARCAL1 and components of the FANCM complex, suggesting novel opportunities for targeted therapies in tumors carrying mutations in these genes. Additionally, we identified robust suppressor interactions between the DCLRE1B gene encoding the nuclease APOLLO and the core non-homologous end joining (NHEJ) genes XRCC4, LIG4, and NHEJ1, suggesting that NHEJ impairs the fitness of APOLLO-deficient cells. This work provides a functional map of the DDR network and demonstrates the power of Cas12a-based screens for identifying synthetic lethal and buffering interactions with therapeutic potential.

genetics↗

bioRxiv2026

SMARCAL1 is a candidate therapeutic target for ALT-positive tumors

A significant subset of tumors, including over 50% of osteosarcomas--an aggressive bone malignancy affecting children, adolescents, and young adults--relies on alternative lengthening of telomeres (ALT), a telomerase-independent, DNA repair-based mechanism for telomere elongation. The overall 5-year survival rate for osteosarcoma patients is [~]65%, underlying the need to develop novel targeted therapies. Through the Cancer Dependency Map, we identify SMARCAL1, a DNA translocase previously shown to remodel stalled replication forks, as a top selective dependency factor in telomerase-negative tumors. Using a panel of ALT-positive and ALT-negative cancer cell lines, as well as osteosarcoma patient-derived xenograft cells, we confirm that ALT-positive cells are uniquely sensitive to the loss of SMARCAL1, whose depletion exacerbates ALT-dependent phenotypes and telomeric DNA damage. Notably, we demonstrate that suppressing ALT abrogates their dependency on SMARCAL1. Mechanistically, we show that SMARCAL1 loss leads to telomeric ssDNA accumulation in ALT-positive cells, dependent in part on DNA repriming mediated by the DNA primase/polymerase PRIMPOL. Moreover, SMARCAL1s ssDNA annealing activity counteracts DNA unwinding by the BLM helicase, limiting telomeric ssDNA accumulation and DNA damage in ALT-positive cells. Importantly, SMARCAL1 depletion induces senescence in ALT-positive cancer cells, rendering them susceptible to treatment with senolytic agents. Together, these findings establish SMARCAL1 as a key regulator of ALT metabolism and highlight SMARCAL1 as a promising therapeutic target for ALT-positive tumors.

cancer biology↗

bioRxiv2025

Distinct mechanisms underlying extrachromosomal telomere DNA generation in ALT cancers

Alternative lengthening of telomeres (ALT) is a telomerase-independent telomere maintenance mechanism observed in 15% of human cancers. A hallmark of ALT cancers is the presence of C-circles, circular single-stranded DNAs (ssDNAs) enriched with cytosine-rich telomere (C-rich, CCCTAA) sequences. G-circles, containing guanosine-rich telomere (G-rich, GGGTTA) ssDNAs, also exist but are much less abundant. Recent studies indicate that excessive displacement of Okazaki fragments during lagging-strand synthesis is a unique feature of ALT telomeres and responsible for generating C-circles/C-rich ssDNAs. However, the distinct characteristics of C-circles compared to G-circles remain unclear. Here, we demonstrate that co-deficiency of the DNA translocases SMARCAL1 and FANCM leads to abundant generation of G-circle/G-rich ssDNAs. These G-rich ssDNAs mainly exist in linear form, ranging in size from 500 to 3000 nucleotides, which differs significantly from the structure and size of C-circle/C-rich ssDNAs. Mechanistically, both C-rich and G-rich ssDNAs originate from BLM/POLD-mediated excessive strand displacement; however, they differ in their origins and initiation mechanisms. Specifically, C-rich ssDNAs arise from lagging daughter strands initiated by the CST complex, whereas G-rich ssDNAs originate from leading daughter strands through RAD51-dependent G-strand synthesis. Our findings propose two distinct mechanisms for generating two different extrachromosomal telomere DNAs, C-and G-circles, during ALT-mediated telomere elongation.

biochemistry↗

bioRxiv2023

CRISPRmap: Sequencing-free optical pooled screens mapping multi-omic phenotypes in cells and tissue

Pooled genetic screens are powerful tools to study gene function in a high-throughput manner. Typically, sequencing-based screens require cell lysis, which limits the examination of critical phenotypes such as cell morphology, protein subcellular localization, and cell-cell/tissue interactions. In contrast, emerging optical pooled screening methods enable the investigation of these spatial phenotypes in response to targeted CRISPR perturbations. In this study, we report a multi-omic optical pooled CRISPR screening method, which we have named CRISPRmap. Our method combines a novel in situ CRISPR guide identifying barcode readout approach with concurrent multiplexed immunofluorescence and in situ RNA detection. CRISPRmap barcodes are detected and read out through combinatorial hybridization of DNA oligos, enhancing barcode detection efficiency, while reducing both dependency on third party proprietary sequencing reagents and assay cost. Notably, we conducted a multi-omic base-editing screen in a breast cancer cell line on core DNA damage repair genes involved in the homologous recombination and Fanconi anemia pathways investigating how nucleotide variants in those genes influence DNA damage signaling and cell cycle regulation following treatment with ionizing radiation or DNA damaging agents commonly used for cancer therapy. Approximately a million cells were profiled with our multi-omic approach, providing a comprehensive phenotypic assessment of the functional consequences of the studied variants. CRISPRmap enabled us to pinpoint likely-pathogenic patient-derived mutations that were previously classified as variants of unknown clinical significance. Furthermore, our approach effectively distinguished barcodes of a pooled library in tumor tissue, and we coupled it with cell-type and molecular phenotyping by cyclic immunofluorescence. Multi-omic spatial analysis of how CRISPR-perturbed cells respond to various environmental cues in the tissue context offers the potential to significantly expand our understanding of tissue biology in both health and disease.

genomics↗

bioRxiv2023

Extrachromosomal Telomeres Derived from Excessive Strand Displacements

Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of C-rich sequences. Despite the fact that extrachromosomal C-rich single-stranded DNAs (ssDNAs), unique to ALT cells, are considered potential precursors of C-circles, their generation process remains undefined. Here, we introduce a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both linear C-rich ssDNAs and C-circles are abundant in the fractions of cytoplasm and nucleoplasm, which supports the idea that linear C-rich ssDNA accumulation may indeed precede C-circle formation. We also found that C-rich ssDNAs originate during Okazaki fragment processing during lagging strand DNA synthesis. The generation of C-rich ssDNA requires CST-PP (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) complex-mediated priming of the C-strand DNA synthesis and subsequent excessive strand displacement of the C-rich strand mediated by the DNA Polymerase delta and the BLM helicase. Our work proposes a new model for the generation of C-rich ssDNAs and C-circles during ALT-mediated telomere elongation.

biochemistry↗

bioRxiv2019

Identification of MCM8IP, an interactor of MCM8-9 and RPA1 that promotes homologous recombination and DNA synthesis in response to DNA damage

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. HR is carried out by a complex network of DNA repair factors. Here we identify C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a novel DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we identify a direct interaction with MCM8-9, a putative helicase complex mutated in Primary Ovarian Insufficiency, that is crucial for MCM8IPs ability to promote resistance to DNA damaging agents. In addition to its association with MCM8-9, MCM8IP also binds directly to RPA1. We show that the interactions of MCM8IP with both MCM8-9 and RPA are required to maintain replication fork progression in response to treatment with crosslinking agents. Collectively, our work identifies MCM8IP as a key regulator of DNA damage-associated DNA synthesis during DNA recombination and replication.

genetics↗