Functional Analysis of the Human Telomeric Protein TRF2

TTAGGG Repeat Binding Factor 2 (TRF2) is a ubiquitous human telomeric protein present at all telomeres throughout the cell cycle. TRF2 has been proposed remodel telomeres into large lasso-like structures named t-loops. Removal of TRF2 from telomeres by expression of a dominant negative allele of TRF2 (TRF2ABAM) led dissolution of these structures and appearance of end-to-end fusions visible in metaphase and anaphase cells. Fusion formation was accompanied by the loss of Goverhang, but not by the loss of double stranded telomeric DNA, which can be documented to persist at the sites of fusions. Fusions were covalent and their formation was dependent on the nonhomologous end-joining (NHEJ) pathway as evidenced by the failure to form fusions in cells lacking Ligase IV. Furthermore, the fusions initiated genomic instability. Expression of TRF2ABAM in primary human cells resulted in induction of senescence. The arrested cells exhibited elevated levels of p53, stabilization of was accompanied by induction of its downstream effectors p21 and Bax. The Rb pathway was also affected, with pRb becoming hypophosphorylated. The importance of both the p53 and the Rb pathways was further evident from the fact that the expression of SV40 Tag alone bypassed the growth arrest. Separate elimination of p53 or Rb function could not do so, and the activity of the ATM PI3 kinase was not necessary the arrest. Expression of a different truncation allele of TRF2, TRF2AB also resulted in senescence but the growth arrest was accompanied by rapid loss of telomeric DNA. In addition, this allele induced an unanticipated chromosome breakage phenotype. Long-term overexpression of full length T R F 2 resulted in the gradual shortening of telomeres, suggesting that TRF2 is a regulator of telomere maintenance. Although it is clear that TRF2 acts through positively regulating the shortening activities at telomeres, it is unknown at this time whether it influences the telomerase pathway. Besides being present at telomeres, TRF2 also appears to be localized to centrosomes. The functional consequences of such localization are not known at this time; however, it is shown that telomere dysfunction can result in genome reduplication

A Genetic Screen Identifies FAN1, a Fanconi Anemia-Associated Nuclease Necessary for DNA Interstrand Crosslink Repair

The Fanconi anemia (FA) pathway is responsible for interstrand crosslink repair. At the heart of this pathway is the FANCI-FAND2 (ID) complex, which, upon ubiquitination by the FA core complex, travels to sites of damage to coordinate repair that includes nucleolytic modification of the DNA urrounding the lesion and translesion synthesis. How the ID complex regulates these events is unknown. Here we describe a shRNA screen that led to the identification of two nucleases necessary for crosslink repair, FAN1 (KIAA1018) and EXDL2. FAN1 colocalizes at sites of DNA damage with the ID complex in a manner dependent on FAN1’s ubiquitin-binding domain (UBZ), the ID complex, and monoubiquitination of FANCD2. FAN1 possesses intrinsic 50 -30 exonuclease activity and endonuclease activity that cleaves nicked and branched structures. We propose that FAN1 is a repair nuclease that is recruited to sites of crosslink damage in part through binding the ubiquitinated ID complex through its UBZ domain

Temporary HMCES-DNA cross-link prevents permanent DNA damage

HMCES, a recently discovered but ancient protein, covalently attaches to damaged single-stranded DNA and shields it from nucleases. Rua-Fernandez and colleagues now show that HMCES catalyzes its own recycling, permitting normal growth and non-mutagenic DNA repair.

Different telomere damage signaling pathways in human and mouse cells

Programmed telomere shortening in human somatic cells is thought to act as a tumor suppressor pathway, limiting the replicative potential of developing tumor cells. Critically short human telomeres induce senescence either by activating p53 or by inducing the p16/RB pathway, and suppression of both pathways is required to suppress senescence of aged human cells. Here we report that removal of TRF2 from human telomeres and the ensuing de-protection of chromosome ends induced immediate premature senescence. Although the telomeric tracts remained intact, the TRF2(ΔBΔM)-induced premature senescence was indistinguishable from replicative senescence and could be mediated by either the p53 or the p16/RB pathway. Telomere de-protection also induced a growth arrest and senescent morphology in mouse cells. However, in this setting the loss of p53 function was sufficient to completely abrogate the arrest, indicating that the p16/RB response to telomere dysfunction is not active in mouse cells. These findings reveal a fundamental difference in telomere damage signaling in human and mouse cells that bears on the use of mouse models for the telomere tumor suppressor pathway

DNA Damage Foci at Dysfunctional Telomeres

AbstractWe report cytologic and genetic data indicating that telomere dysfunction induces a DNA damage response in mammalian cells. Dysfunctional, uncapped telomeres, created through inhibition of TRF2, became associated with DNA damage response factors, such as 53BP1, γ-H2AX, Rad17, ATM, and Mre11. We refer to the domain of telomere-associated DNA damage factors as a Telomere Dysfunction-Induced Focus (TIF). The accumulation of 53BP1 on uncapped telomeres was reduced in the presence of the PI3 kinase inhibitors caffeine and wortmannin, which affect ATM, ATR, and DNA-PK. By contrast, Mre11 TIFs were resistant to caffeine, consistent with previous findings on the Mre11 response to ionizing radiation. A-T cells had a diminished 53BP1 TIF response, indicating that the ATM kinase is a major transducer of this pathway. However, in the absence of ATM, TRF2 inhibition still induced TIFs and senescence, pointing to a second ATM-independent pathway. We conclude that the cellular response to telomere dysfunction is governed by proteins that also control the DNA damage response. TIFs represent a new tool for evaluating telomere status in normal and malignant cells suspected of harboring dysfunctional telomeres. Furthermore, induction of TIFs through TRF2 inhibition provides an opportunity to study the DNA damage response within the context of well-defined, physically marked lesions

Współnauczanie i jego znaczenie dla procesu edukacji. Przegląd badań

Niniejszy artykuł jest próbą syntezy wyników badań nad znaczeniem współnauczania dla przebiegu procesu edukacji. W pierwszej części tekstu zdefiniowano pojęcie współnauczania i dokonano klasyfikacji obszarów, w ramach których analizowane jest współnauczanie. Następnie opisano istniejące modele współnauczania, czynniki, od których może zależeć jakość współpracy między nauczycielami, a także korzyści płynące z takiego sposobu współpracy dla pedagogów. Na końcu omówiono znaczenie współnauczania dla uczniów zarówno w aspekcie ich rozwoju poznawczego, jak i społecznego.This manuscript attempts to synthesise results of research on importance of co-teaching for the process of education. In the first part of the text we defined co-teaching and we classified areas, in which co-teaching is analysed. Further, we described existing models of co-teaching, potential determinants of cooperation between teachers, as well as benefits of such way of collaboration. At the end we discussed importance of co-teaching for students – in the case of cognitive, as well as social functioning

Human GEN1 and the SLX4-Associated Nucleases MUS81 and SLX1 Are Essential for the Resolution of Replication-Induced Holliday Junctions

Holliday junctions (HJs), the DNA intermediates of homologous recombination, need to be faithfully processed in order to preserve genome integrity. In human cells, the BLM helicase complex promotes nonnucleolytic dissolution of double HJs. In vitro, HJs may be nucleolytically processed by MUS81-EME1, GEN1, and SLX4-SLX1. Here, we exploit human SLX4-null cells to examine the requirements for HJ resolution in vivo. Lack of BLM and SLX4 or GEN1 and SLX4 is synthetically lethal in the absence of exogenous DNA damage, and lethality is a consequence of dysfunctional mitosis proceeding in the presence of unprocessed HJs. Thus, GEN1 activity cannot be substituted for the SLX4-associated nucleases, and one of the HJ resolvase activities, either of those associated with SLX4 or with GEN1, is required for cell viability, even in the presence of BLM. In vivo HJ resolution depends on both SLX4-associated MUS81-EME1 and SLX1, suggesting that they are acting in concert in the context of SLX4

A genome-wide homologous recombination screen identifies the RNA-binding protein RBMX as a component of the DNA-damage response

Repair of DNA double-strand breaks is critical to genomic stability and the prevention of developmental disorders and cancer. A central pathway for this repair is homologous recombination (HR). Most knowledge of HR is derived from work in prokaryotic and eukaryotic model organisms. We carried out a genome-wide siRNA-based screen in human cells. Among positive regulators of HR we identified networks of DNA-damage-response and pre-mRNA-processing proteins, and among negative regulators we identified a phosphatase network. Three candidate proteins localized to DNA lesions, including RBMX, a heterogeneous nuclear ribonucleoprotein that has a role in alternative splicing. RBMX accumulated at DNA lesions through multiple domains in a poly(ADP-ribose) polymerase 1-dependent manner and promoted HR by facilitating proper BRCA2 expression. Our screen also revealed that off-target depletion of RAD51 is a common source of RNAi false positives, raising a cautionary note for siRNA screens and RNAi-based studies of HR

Treatment of Fanconi Anemia–Associated Head and Neck Cancer: Opportunities to Improve Outcomes

Fanconi anemia, the most frequent genetic cause of bone marrow failure, is characterized by an extreme predilection toward multiple malignancies, including a greater than 500-fold incidence of head and neck squamous cell carcinoma (HNSCC) relative to the general population. Fanconi anemia-associated HNSCC and esophageal SCC (FA-HNSCC) often present at advanced stages with poor survival. Surgical resection remains the primary treatment for FA-HNSCC, and there is often great reluctance to administer systemic agents and/or radiotherapy to these patients given their susceptibility to DNA damage. The paucity of FA-HNSCC case reports limits evidence-based management, and such cases have not been analyzed collectively in detail. We present a systematic review of FA-HNSCC treatments reported from 1966 to 2020, defining a cohort of 119 patients with FA-HNSCC including 16 esophageal SCCs (131 total primary tumors), who were treated with surgery, radiotherapy, systemic therapy (including cytotoxic agents, EGFR inhibitors, or immune checkpoint inhibitors), or a combination of modalities. We summarize the clinical responses and regimen-associated toxicities by treatment modality. The collective evidence suggests that when possible, surgical resection with curative intent should remain the primary treatment modality for FA-HNSCC. Radiation can be administered with acceptable toxicity in the majority of cases, including patients who have undergone stem cell transplantation. Although there is little justification for cytotoxic chemotherapy, EGFR inhibitors and tyrosine kinase inhibitors may be both safe and effective. Immunotherapy may also be considered. Most oncologists have little personal experience with FA-HNSCC. This review is intended as a comprehensive resource for clinicians