T loops and telomeric G-quadruplex (G4) DNA structures pose a potential threat to genome stability and must be dismantled to permit efficient telomere replication. Here we implicate the helicase RTEL1 in the removal of telomeric DNA secondary structures, which is essential for preventing telomere fragility and loss. In the absence of RTEL1, T loops are inappropriately resolved by the SLX4 nuclease complex, resulting in loss of the telomere as a circle. Depleting SLX4 or blocking DNA replication abolished telomere circles (TCs) and rescued telomere loss in RTEL1(-/-) cells but failed to suppress telomere fragility. Conversely, stabilization of telomeric G4-DNA or loss of BLM dramatically enhanced telomere fragility in RTEL1-deficient cells but had no impact on TC formation or telomere loss. We propose that RTEL1 performs two distinct functions at telomeres: it disassembles T loops and also counteracts telomeric G4-DNA structures, which together ensure the dynamics and stability of the telomere.
Natural killer/T-cell lymphoma (NKTCL) is a malignant proliferation of CD56(+) and cytoCD3(+) lymphocytes with aggressive clinical course, which is prevalent in Asian and South American populations. The molecular pathogenesis of NKTCL has largely remained elusive. We identified somatic gene mutations in 25 people with NKTCL by whole-exome sequencing and confirmed them in an extended validation group of 80 people by targeted sequencing. Recurrent mutations were most frequently located in the RNA helicase gene DDX3X (21/105 subjects, 20.0%), tumor suppressors (TP53 and MGA), JAK-STAT-pathway molecules (STAT3 and STAT5B) and epigenetic modifiers (MLL2, ARID1A, EP300 and ASXL3). As compared to wild-type protein, DDX3X mutants exhibited decreased RNA-unwinding activity, loss of suppressive effects on cell-cycle progression in NK cells and transcriptional activation of NF-κB and MAPK pathways. Clinically, patients with DDX3X mutations presented a poor prognosis. Our work thus contributes to the understanding of the disease mechanism of NKTCL.
C hronic stalling of DNA replication forks by DNA damage such as UV irradiation, ionizing irradiation, chemicals, and reactive cellular metabolites impedes the progression of the cell cycle and eventually causes cell death. To circumvent such situations, cells have evolved the postreplication repair (PRR) pathway that bypasses DNA lesions to resolve stalled forks without removing the actual damage (1). In budding yeast Saccharomyces cerevisiae, PRR is carried out by 2 distinct pathways: translesion synthesis (TLS) and template switching (TS) (Fig. 1A). TLS uses multiple low-fidelity TLS polymerases to incorporate nucleotides across DNA lesions (2, 3). Switching from replicative polymerases ␦ or to TLS polymerases is promoted through the interaction between monoubiquitinated PCNA at lysine 164 (K164) and a ubiquitin-binding motif in TLS polymerases-a mechanism conserved from the budding yeast to human. The monoubiquitination of PCNA at K164 requires the RING-type ubiquitin ligase Rad18 (E3) and the ubiquitinconjugating enzyme Rad6 (E2).The TS pathway bypasses DNA damage by switching a stalled replicating end to the nascent daughter strand of the sister chromatid (1, 4). This pathway involves a lysine 63 (K63)-linked polyubiquitin chain that is further added onto the monoubiquitinated PCNA by Rad5 (E3) along with the Ubc13-Mms2 (E2 and E2 variant, respectively) heterodimer complex (Fig. 1 A). Distinct from the K48-linked polyubiquitination leading to protein degradation, the K63-linked polyubiquitination of PCNA is thought to promote TS in a proteasome-independent manner (5).The importance of the TLS pathway in the suppression of mammalian tumorigenesis emerged with the identification of a mutation in TLS polymerase in patients with the variant form of xeroderma pigmentosum and from studies with mouse models (6, 7). Despite the presence of UBC13 and MMS2 homologues in humans, the importance of the TS pathway is less clear in mammals because K63-linked polyubiquitination of PCNA, a hallmark event for the TS pathway, had not been observed until recently (8-10). We recently identified human SHPRH, which possesses SWI2/SNF2 and RING domains with similar architecture to the yeast Rad5 as a functional homologue of yeast Rad5 (9). Specifically, we demonstrated the in vivo activity of SHPRH in promoting a K63-linked polyubiquitination of PCNA as well as physical interactions of SHPRH with PCNA, RAD18, and UBC13. Depletion of SHPRH increases genomic instability after genotoxic stress. Consistent with our work, another study also demonstrated that SHPRH could polyubiquitinate PCNA in vitro (11).In the present study, we demonstrated that ectopic expression of HLTF/SMARCA3/RUSH/HIP116/Zbu1 (hereafter, HLTF) enhanced PCNA polyubiquitination in vivo. Depletion of SHPRH or HLTF significantly reduced polyubiquitination of chromatin-bound PCNA upon treatment of cells with DNA-damaging agents that cause stalled DNA replication forks. Furthermore, Hltf-deficient mouse embryonic fibroblasts (MEFs) showed elevated chromosome breaks an...
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