Biomedical subjects

Lee Zou

Publications and source records attributed to Lee Zou.

13 recordsLinked to original sources

PubMed2026

FANCM is required for the PAX3::FOXO1-driven oncogenic program in rhabdomyosarcoma.

Many cancers are driven by mutationally altered transcription factors (TFs) that rewire cells to an oncogenic state. Cells must activate specific mechanisms to tolerate the burden of oncogenic TF activity. To define such mechanisms, we focused on a canonical oncogenic fusion protein-driven cancer, alveolar rhabdomyosarcoma (ARMS), where the PAX3::FOXO1 fusion protein hyperactivates and mislocalizes PAX3 and FOXO1 TF functions. Employing sequential functional genomic CRISPR-Cas9 screens, we identified FANCM, a DNA translocase in the Fanconi anemia pathway, as a selective dependency in PAX3::FOXO1+ ARMS. FANCM loss reduces fusion protein levels, induces myogenic differentiation, and disrupts the PAX3::FOXO1 transcriptional program, thereby halting oncogenic proliferation. Mechanistically, FANCM depletion exacerbates replication stress (RS) and DNA damage signaling, with chromatin-associated RS enriched at PAX3::FOXO1 target gene loci, resulting in selective downregulation of the oncogenic program. CRISPR exon-tiling screens prioritized FANCM's helicase and DNA-binding domains as essential for this dependency, linking FANCM-mediated replication fork binding to sustained oncogenesis.

ARMS↗

PubMed2026

Suppression of transcription-replication conflicts by sequence-coordinated actions of TRDMT1 and MutLα.

TRDMT1 is an RNA methyltransferase that catalyzes 5-methylcytosine (m5C) formation in R-loops to promote transcription-coupled homologous recombination (TC-HR). Although TRDMT1 inhibition selectively kills BRCA1-deficient cancer cells, broader cancer dependencies on TRDMT1 remain unclear. Here, a TRDMT1 inhibitor (TRDMT1i) sensitivity screen across a large panel of cancer cell lines identifies loss of MLH1 or PMS2, two components of the MutLα mismatch repair (MMR) complex frequently inactivated in tumors, as key determinants of TRDMT1 dependency. In contrast, MutLβ and MutSα/β are dispensable for TRDMT1i resistance, revealing a unique MMR-independent function of MutLα. Mechanistically, TRDMT1 and MutLα independently recognize DNA-RNA hybrids and cooperatively suppress co-transcriptional R-loops genome-wide in undamaged cells, with m5C directing pathway choice. Furthermore, MutLα suppresses R-loops through its ATPase and endonuclease activities and through recruitment of EXO1. Combined loss of TRDMT1 and MLH1 causes extensive R-loop accumulation and transcription replication conflicts (TRCs), impairing replication fork progression, inducing DNA damage, and driving apoptosis-mediated synthetic lethality. Importantly, TRDMT1i suppresses growth of MLH1-deficient tumors by inducing TRCs in vivo, suggesting a potential therapeutic strategy for targeting MutLα-deficient tumors. These studies not only expand our understanding of cancer dependency on TRDMT1, but also identify a promising strategy to exploit TRCs in cancer therapy.

Humans↗

PubMed2026

BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance.

Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.

Humans↗

PubMed2006

Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress.

The ATR-mediated checkpoint is not only critical for responding to genotoxic stress but also essential for cell proliferation. The RFC-related checkpoint protein Rad17, a phosphorylation substrate of ATR, is critical for ATR-mediated checkpoint signaling and cell survival. Here, we show that phosphorylation of Rad17 by ATR is important for genomic stability and restraint of S phase but is not essential for cell survival. The phosphomutant Rad17AA exhibits distinct defects in hydroxyurea- (HU) and ultraviolet- (UV) induced Chk1 activation, indicating that separate Rad17 functions are required differently in response to different types of replication interference. Although cells expressing Rad17AA can initiate Chk1 phosphorylation after HU treatment, they fail to sustain Chk1 phosphorylation after withdrawal of HU and are profoundly sensitive to HU. Importantly, we found that phosphorylated Rad17 interacts with Claspin and regulates its phosphorylation. These findings reveal a phosphorylation-dependent function of Rad17 in an ATR-Rad17-Claspin-Chk1-signaling cascade that responds to specific replication stress.

Adaptor Proteins, Signal Transducing↗

PubMed2006

ATRIP associates with replication protein A-coated ssDNA through multiple interactions.

The ATR (ATM- and rad3-related)-mediated checkpoint pathway has a crucial role in regulating the cellular responses to DNA damage and DNA-replication stress. ATRIP (ATR-interacting protein), the regulatory partner of ATR, binds directly to replication protein A (RPA)-coated ssDNA and enables the ATR-ATRIP complex to recognize this DNA damage-induced structure. Here, we show that ATRIP associates with RPA-ssDNA through multiple interactions. Two major RPA-ssDNA-interacting domains of ATRIP were mapped to the regions flanking the conserved coiled-coil domain. In contrast to a recent article, we found that ATRIP mutants lacking the N terminus retained the ability to bind to RPA-ssDNA, suggesting that the multiple interactions between ATRIP and RPA-ssDNA may function redundantly in the recruitment of ATR-ATRIP. Unexpectedly, one internal region of ATRIP exhibited affinity to ssDNA, suggesting that ATRIP may interact with ssDNA in the ATRIP-RPA-ssDNA complex. Also, the N terminus of ATRIP associated with RPA-ssDNA in two distinct ways, indicating a dynamic and regulated association between ATRIP and RPA-ssDNA.

Adaptor Proteins, Signal Transducing↗

PubMed2006

Recruitment of ATR-ATRIP, Rad17, and 9-1-1 complexes to DNA damage.

The ATR (ataxia-telangiectasia mutated and rad3-related)-ATRIP (ATR-interacting protein) kinase complex plays a central role in the checkpoint responses to a variety of types of DNA damage, especially those interfering with DNA replication. The checkpoint-signaling pathway activated by ATR-ATRIP regulates and coordinates cell-cycle progression, DNA replication, DNA repair, and many other cellular processes critical for genomic stability. Upon DNA damage or DNA replication interference, ATR-ATRIP and two of its key regulators, the Rad17 and the 9-1-1 complexes, are localized to sites of DNA damage and stalled replication forks. Recent biochemical and cell biological studies have revealed that RPA-coated single-stranded DNA, a common structure generated at sites of DNA damage and stalled replication forks, plays crucial roles in the recruitment of ATR-ATRIP, Rad17, and 9-1-1 complexes. The recruitment of ATR-ATRIP and its regulators to DNA damage is a key step for the recognition of DNA damage by the checkpoint, and is likely important for the regulation of ATR activity and/or function in response to DNA damage. The methods used to characterize the DNA association of ATR-ATRIP, Rad17, and 9-1-1 complexes have laid a foundation for further biochemical studies, which may ultimately lead us to understand the molecular mechanisms by which ATR-ATRIP monitors and protects genomic integrity.

Cell Cycle Proteins↗

PubMed2006

Checkpoint and coordinated cellular responses to DNA damage.

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. The activation of checkpoints not only attenuates cell cycle progression, but also facilitates DNA repair and recovery of faulty replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. It has become increasingly clear that the activation and signaling of the checkpoint are intimately linked to the cellular processes directly involved in chromosomal metabolism, such as DNA replication and DNA repair. Thus, the checkpoint pathway is not just a surveillance system that monitors genomic integrity and regulates cell proliferation, but also an integral part of the processes that work directly on chromosomes to maintain genomic stability. In this article, we discuss the current models of DNA damage and replication checkpoints, and highlight recent advances in the field.

Animals↗

PubMed2005

Sensing, signaling, and responding to DNA damage: organization of the checkpoint pathways in mammalian cells.

The DNA damage and replication checkpoints are signaling mechanisms that regulate and coordinate cellular responses to genotoxic conditions. Unlike typical signal transduction mechanisms that respond to one or a few stimuli, checkpoints can be activated by a broad spectrum of extrinsically or intrinsically derived DNA damage or replication interference. Recent investigations have shed light on how the damage and replication checkpoints are able to respond to such diverse stimuli. The activation of checkpoints not only attenuates cell cycle progression but also facilitates DNA repair and recovery of faltered replication forks, thereby preventing DNA lesions from being converted to inheritable mutations. Recently, more checkpoint targets from the cell cycle and DNA replication apparatus have been identified, revealing the increasing complexity of the checkpoint control of the cell cycle. In this article, we discuss current models of the DNA damage and replication checkpoints and highlight recent advances in the field.

Animals↗

PubMed2003

Replication protein A-mediated recruitment and activation of Rad17 complexes.

The human Rad17-Rfc2-5 and Rad9-Rad1-Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways. In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells. However, the DNA structures recognized by the Rad17-Rfc2-5 complex during the damage response have not been defined. Here, we show that replication protein A (RPA) stimulates the binding of the Rad17-Rfc2-5 complex to single-stranded DNA (ssDNA), primed ssDNA, and a gapped DNA structure. Furthermore, RPA facilitates the recruitment of the Rad9-Rad1-Hus1 complex by the Rad17-Rfc2-5 complex to primed and gapped DNA structures in vitro. These findings suggest that RPA-coated ssDNA is an important part of the structures recognized by the Rad17-Rfc2-5 complex. Unlike replication factor C (RFC), which uses the 3' primer/template junction to recruit proliferating cell nuclear antigen (PCNA), the Rad17-Rfc2-5 complex can use both the 5' and the 3' primer/template junctions to recruit the Rad9-Rad1-Hus1 complex, and it shows a preference for gapped DNA structures. These results explain how the Rad17-Rfc2-5 complex senses DNA damage and DNA replication stress to initiate checkpoint signaling.

Cell Cycle Proteins↗

PubMed2003

Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.

The function of the ATR (ataxia-telangiectasia mutated- and Rad3-related)-ATRIP (ATR-interacting protein) protein kinase complex is crucial for the cellular response to replication stress and DNA damage. Here, we show that replication protein A (RPA), a protein complex that associates with single-stranded DNA (ssDNA), is required for the recruitment of ATR to sites of DNA damage and for ATR-mediated Chk1 activation in human cells. In vitro, RPA stimulates the binding of ATRIP to ssDNA. The binding of ATRIP to RPA-coated ssDNA enables the ATR-ATRIP complex to associate with DNA and stimulates phosphorylation of the Rad17 protein that is bound to DNA. Furthermore, Ddc2, the budding yeast homolog of ATRIP, is specifically recruited to double-strand DNA breaks in an RPA-dependent manner. A checkpoint-deficient mutant of RPA, rfa1-t11, is defective for recruiting Ddc2 to ssDNA both in vivo and in vitro. Our data suggest that RPA-coated ssDNA is the critical structure at sites of DNA damage that recruits the ATR-ATRIP complex and facilitates its recognition of substrates for phosphorylation and the initiation of checkpoint signaling.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Genomic instability and endoreduplication triggered by RAD17 deletion.

Cell cycle checkpoints are critical for genomic stability. Rad17, a component of the checkpoint clamp loader complex (Rad17/Rfc2-5), is required for the response to DNA damage and replication stress. To explore the role of Rad17 in the maintenance of genomic integrity, we established somatic conditional alleles of RAD17 in human cells. We find that RAD17 is not only important for the Atr-mediated checkpoint but is also essential for cell viability. Cells lacking RAD17 exhibited acute chromosomal aberrations and underwent endoreduplication at a high rate. Therefore, RAD17 links the checkpoint to ploidy control and is essential for the maintenance of chromosomal stability.

Blotting, Southern↗

PubMed2002

Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.

Cells respond to DNA damage by activating a network of signaling pathways that control cell cycle progression and DNA repair. Genetic studies in yeast suggested that several checkpoint proteins, including the RFC-related Rad17 protein, and the PCNA-related Rad1-Rad9-Hus1 protein complex might function as sensors of DNA damage. In this study, we show that the human Rad17 protein recruits the Rad9 protein complex onto chromatin after damage. Rad17 binds to chromatin prior to damage and is phosphorylated by ATR on chromatin after damage but Rad17's phosphorylation is not required for Rad9 loading onto chromatin. The chromatin associations of Rad17 and ATR are largely independent, which suggests that they localize to DNA damage independently. Furthermore, the phosphorylation of Rad17 requires Hus1, suggesting that the Rad1-Rad9-Hus1 complex recruited by Rad17 enables ATR to recognize its substrates. Our data are consistent with a model in which multiple checkpoint protein complexes localize to sites of DNA damage independently and interact to trigger the checkpoint-signaling cascade.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2002

Checking on the fork: the DNA-replication stress-response pathway.

To ensure the fidelity of DNA replication, cells activate a stress-response pathway when DNA replication is perturbed. This pathway regulates not only progress through the cell cycle but also transcription, apoptosis, DNA repair/recombination and DNA replication itself. Mounting evidence has suggested that this pathway is important for the maintenance of genomic integrity. Here, we discuss recent findings about how this pathway is activated by replication stress and how it regulates the DNA-replication machinery to alleviate the stress.

Animals↗