Biology subjects

Lue, N.

Publications and source records attributed to Lue, N..

7 recordsLinked to original sources

bioRxiv2026

CST-Polα/primase, backed up by Rif1, protects telomeric ss-dsDNA junctions from damage recognition and resection

The yeast Cdc13-Stn1-Ten1 (CST) complex is critical for telomere length regulation and telomere protection. Recent studies further implicate Pol/primase (PP) as an essential partner of CST in executing these functions, but the mechanistic bases remain poorly understood. Using the pathogenic yeast Nakaseomyces glabratus, a model amenable to mechanistic dissection of CST-PP, we analyzed the protection mechanisms of CST-PP by combining mutations that disrupt individual subunits or complex assembly with mutations in DNA damage response (DDR) and DNA repair factors. The complete loss of Cdc13 - which eliminates the CST-PP assembly - results in telomere damage recognition involving Rad9, Rad24 and Mec3, as well as aberrant resection mediated by Exo1 and Sgs1. In contrast, disrupting the interaction between CST and PP leads to proliferation defects triggered primarily by Exo1, implying a more selective protection function for PP. The involvement of the DDR clamp loader and clamp together with Exo1 points to ss-dsDNA junctions with recessed 5' ends as a crucial lesion in the absence of Cdc13. Further analysis implicates RNase H1 and H2 in promoting DNA damage, possibly by exposing such junctions. Based on similarities between Cdc13 and POT1, a telomere protein previously implicated in junctional protection, we modeled Cdc13 binding to a ss-dsDNA junction and showed that point mutations at the proposed interaction site impaired telomere protection and altered the distribution of the telomere C-strand 5' ends in vivo. We further demonstrate that Rif1, another telomere protein with junctional binding activity, supports the protection function of CST-PP. Together, our findings illuminate the molecular basis of telomere DNA damage and of CST-PP mediated telomere protection.

molecular biology↗

bioRxiv2026

PAXIP1-PAGR1 directs cohesin recruitment during break-induced telomere repair

Cohesin is a conserved multiprotein complex (SMC1, SMC3, RAD21, and either STAG1 or STAG2) that organizes three-dimensional genome architecture and regulates chromosome segregation, gene expression, and DNA damage repair (1-4). Following double-strand breaks (DSBs), cohesin is recruited to sites of DNA damage - a process considered essential for efficient homologous recombination (5-15). Yet how DSB signaling elicits cohesin recruitment and subsequent cohesion establishment remains poorly understood. Here we show that telomere replication stress activates de novo STAG2-cohesin loading, thereby promoting break-induced telomeric DNA repair. We demonstrate that this DNA break-elicited cohesin recruitment is strictly controlled by the BRCT domain-containing DNA damage recognition factor PAXIP1 and its functional partner PAGR1. Cryo-electron microscopy structure reveals that PAGR1, together with PAXIP1, physically binds to a composite interface formed by the STAG2-RAD21 cohesin subcomplex. Complementary mutational and biochemical analyses define the molecular basis of this interaction and establish its essential role in break-induced cohesion establishment. Furthermore, we show that PAXIP1-PAGR1-enacted STAG2-cohesin recruitment complements with the PML body-associated pathway in orchestrating break-induced alternative lengthening of telomeres (ALT). Concurrent depletion of PML together with PAXIP1, PAGR1 or STAG2 disrupts ALT-mediated telomere maintenance, leading to end-to-end chromosomal fusion and mitotic cell death. Collectively, these findings uncover a distinctive molecular mechanism through which DSB signaling directs de novo cohesion establishment, and highlight its critical importance in break-induced telomere repair.

molecular biology↗

bioRxiv2024

The yeast CST and Polα/primase complexes act in concert to ensure proper telomere maintenance and protection

Pol/primase, the polymerase that initiates DNA synthesis at replication origins, also completes the task of genome duplication by synthesizing the telomere C-strand under the control of the CST complex. Using cryo-EM structures of the human CST-Pol/primase-DNA complex as guides in conjunction with AlphaFold modeling, we identified structural elements in yeast CST and Pol/primase that promote complex formation. Mutating these structures in Candida glabrata Stn1, Ten1, Pri1 and Pri2 abrogated the stimulatory activity of CST on Pol/primase in vitro, supporting the functional relevance of the physical contacts in cryo-EM structures as well as the conservation of mechanisms between yeast and humans. Introducing these mutations into C. glabrata yielded two distinct groups of mutants. One group exhibited progressive, telomerase-dependent telomere elongation without evidence of DNA damage. The other manifested slow growth, telomere length heterogeneity, ssDNA accumulation and elevated C-circles, which are indicative of telomere deprotection. These telomere deprotection phenotypes are altered or suppressed by mutations in multiple DDR and DNA repair factors. We conclude that in yeast, the telomerase inhibition and telomere protection function previously ascribed to the CST complex are mediated jointly by both CST and Pol/primase, highlighting the critical importance of a replicative DNA polymerase in telomere regulation.

molecular biology↗

bioRxiv2023

Ustilago maydis Trf2 ensures genome stability by antagonizing Blm-mediated telomere recombination: optimizing DNA repair factor activity through opposing regulations

TRF2 is an essential and conserved double-strand telomere binding protein that stabilizes chromosome ends by suppressing DNA damage response and aberrant DNA repair. Herein we investigated the mechanisms and functions of the Trf2 ortholog in the basidiomycete fungus Ustilago maydis, which manifests strong resemblances to metazoans with regards to the telomere and DNA repair machinery. We showed that UmTrf2 binds to Blm in vitro and inhibits Blm-mediated unwinding of telomeric DNA substrates. Consistent with a similar inhibitory activity in vivo, over-expression of Trf2 induces telomere shortening, just like deletion of blm, which is required for efficient telomere replication. While the loss of Trf2 engenders growth arrest and multiple telomere aberrations, these defects are fully suppressed by the concurrent deletion of blm or mre11 (but not other DNA repair factors). Over-expression of Blm alone triggers aberrant telomere recombination and the accumulation of aberrant telomere structures, which are blocked by concurrent Trf2 over-expression. Together, these findings highlight the suppression of Blm as a key protective mechanism of Trf2. Notably, U. maydis harbors another double-strand telomere-binding protein (Tay1), which promotes Blm activity to ensure efficient replication. We found that deletion of tay1 partially suppresses the telomere aberration of Trf2-depleted cells. Our results thus point to opposing regulation of Blm helicase by telomere proteins as a strategy for optimizing both telomere maintenance and protection. We also show that aberrant transcription of both telomere G- and C-strand is a recurrent phenotype of telomere mutants, underscoring another potential similarity between double strand breaks and de-protected telomeres. Author SummaryThe ends of linear chromosomes are protected from abnormal repair by a collection of telomere proteins. One protein that plays an especially prominent role is TRF2, which binds to double-stranded telomere repeats. In this study, we analyzed the mechanisms and functions of Trf2 in a yeast-like fungus named Ustilago maydis, which manifests a high degree of similarity to animal cells with respect to telomere regulation. We showed that Trf2 binds directly to a conserved DNA helicase called Blm and inhibits the ability of Blm to unwind telomeric DNA in a purified, cell-free reaction. We also used over-expression and depletion of either Trf2 or Blm or both to demonstrate an inhibitory effect of Trf2 on Blm function in vivo. For example, depletion of Trf2 triggers Blm-dependent telomere aberrations and cell death. Interestingly, another double-strand telomere binding protein named Tay1 was found to stimulate Blm activity to promote telomere replication. Together, our results indicate that U. maydis optimizes Blm function through opposing regulation of its activity via distinct telomere proteins. We also detected high levels of abnormal transcripts that correspond to both strands of telomeres in a variety of telomere mutants, suggesting that de-protected telomeres are permissive substrates for the transcription apparatus.

molecular biology↗

bioRxiv2023

Histone demethylase KDM2A is a selective vulnerability of cancers relying on alternative telomere maintenance

Telomere length maintenance is essential for cellular immortalization and tumorigenesis. 5% - 10% of human cancers rely on a recombination-based mechanism termed alternative lengthening of telomeres (ALT) to sustain their replicative immortality, yet there are currently no targeted therapies. Through CRISPR/Cas9-based genetic screens in an ALT-immortalized isogenic cellular model, here we identify histone lysine demethylase KDM2A as a molecular vulnerability selectively for cells contingent on ALT-dependent telomere maintenance. Mechanistically, we demonstrate that KDM2A is required for dissolution of the ALT-specific telomere clusters following homology-directed telomere DNA synthesis. We show that KDM2A promotes de-clustering of ALT multitelomeres through facilitating isopeptidase SENP6-mediated SUMO deconjugation at telomeres. Inactivation of KDM2A or SENP6 impairs post-recombination telomere de-SUMOylation and thus dissolution of ALT telomere clusters, leading to gross chromosome missegregation and mitotic cell death. These findings together establish KDM2A as a selective molecular vulnerability and a promising drug target for ALT-dependent cancers.

cancer biology↗

bioRxiv2021

Analysis of Ustilago maydis Pot1 reveals context-dependent, dichotomous regulation of homology-directed DNA repair factors by a telomere protein

The telomere G-strand binding protein Pot1 plays multifaceted roles in telomere maintenance and protection. We examined the structure and activities of Pot1 in Ustilago maydis, a fungal model that recapitulates key features of mammalian telomere regulation. Compared to the well-characterized primate and fission yeast Pot1 orthologs, UmPot1 harbors an extra N-terminal OB-fold domain (OB-N), which was recently shown to be present in most metazoans. UmPot1 binds directly to Rad51 and regulates the latters strand exchange activity. Deleting the OB-N domain, which is implicated in Rad51-binding, caused telomere shortening, suggesting that Pot1-Rad51 interaction facilitates telomere replication. Depleting Pot1 through transcriptional repression triggered growth arrest as well as rampant recombination, leading to multiple telomere aberrations. In addition, telomere repeat RNAs transcribed from both the G- and C-strand were dramatically up-regulated, and this was accompanied by elevated levels of telomere RNA-DNA hybrids. Telomere abnormalities of pot1-deficient cells were suppressed, and cell viability was restored by the deletion of genes encoding Rad51 or Brh2 (the BRCA2 ortholog), indicating that homology-directed repair (HDR) proteins are key mediators of telomere aberrations and cellular toxicity. Together, these observations underscore the complex physical and functional interactions between Pot1 and DNA repair factors, leading to context-dependent and dichotomous effects of HDR proteins on telomere maintenance and protection.

molecular biology↗

bioRxiv2021

Reciprocal impacts of telomerase activity and tumor cell differentiation in neuroblastoma tumor biology

Telomere maintenance and tumor cell differentiation have been separately implicated in neuroblastoma malignancy. Their mechanistic connection is unclear. We analyzed neuroblastoma cell lines and morphologic subclones representing the adrenergic (ADRN) and mesenchymal (MES) differentiation states and uncovered sharp differences in their telomere protein and telomerase activity levels. Pharmacologic conversion of ADRN into MES cells elicited consistent and robust changes in the expression of telomere-related proteins. Conversely, stringent down-regulation of telomerase activity triggers the differentiation of ADRN into MES cells, which was reversible upon telomerase upregulation. Interestingly, the MES differentiation state is associated with elevated levels of innate immunity factors, including key components of the DNA-sensing pathway. Accordingly, MES but not ADRN cells can mount a robust response to viral infections in vitro. A gene expression signature based on telomere and cell lineage-related factors can cluster neuroblastoma tumor samples into predominantly ADRN or MES-like groups, with distinct clinical outcomes. Our findings establish a novel mechanistic connection between telomere and differentiation and suggest that manipulating telomeres may suppress malignancy not only by limiting the tumor growth potential but also by inducing tumor cell differentiation and altering its immunogenicity.

cancer biology↗