A central function of telomeres is to prevent chromosome ends from being recognized as DNA double‐strand breaks (DSBs). Several proteins involved in processing DSBs associate with telomeres, but the ...roles of these factors at telomeres are largely unknown. To investigate whether the Mre11/Rad50/Nbs1 (MRN) complex is involved in the generation of proper 3′ G‐overhangs at human telomere ends, we used RNA interference to decrease expression of MRN and analysed their effects. Reduction of MRN resulted in a transient shortening of G‐overhang length in telomerase‐positive cells. The terminal nucleotides of both C‐ and G‐rich strands remain unaltered in Mre11‐diminished cells, indicating that MRN is not responsible for specifying the final end‐processing event. The reduction in overhang length was not seen in telomerase‐negative cells, but was observed after the expression of exogenous telomerase, which suggested that the MRN complex might be involved in the recruitment or action of telomerase.
Abstract only
Following telomere de‐protection chromosome ends are fused either by a Ligase 4‐dependent classical NHEJ process (C‐NHEJ) or using an alternative NHEJ pathway (alt‐NHEJ) mediated by ...Ligase 3 and PARP1. We recently investigated the differences in sequence fidelity between the two end‐joining reactions, and identified Polymerase theta (Polθ; encoded by
Polq
) as a critical alt‐NHEJ factor that introduces random nucleotides at fusion junctions.
Polq
inhibition suppresses alt‐NHEJ at telomeres that have lost shelterin protection, and blocks translocations at non‐telomeric loci. In parallel, loss of
Polq
results in increased rates of homology directed repair (HDR). Interestingly, depleting
Polq
in cells carrying mutations in the breast‐cancer susceptibility genes (Brca1 or Brca2
)
exacerbates chromosomal aberrancies and reduces cellular survival, suggesting that Polθ‐mediated alt‐NHEJ compensates when HDR is impaired.
Polθ is an atypical A‐family DNA polymerase with an N‐terminal helicase‐like domain, a large central domain harboring a Rad51 interaction motif, and a C‐terminal polymerase domain capable of extending DNA strands from mismatched or even unmatched termini.
In vitro
experiments identified an evolutionarily conserved loop in the polymerase domain that is essential for synapsing DNA ends during end joining. To address the mechanism by which this low‐fidelity polymerase orchestrates double‐strand break repair we employed CRISPR/Cas9 gene editing and established cell lines with key Polθ mutations (PolqΔhelicase, PolqΔRAD51, PolqΔpolymerase, and PolqΔloop). Here we report on the impact of these mutations on Polθ function in promoting alt‐NHEJ and suppressing HDR, and address the importance of Polθ‐mediated alt‐NHEJ in survival of HDR defective cells.
The fidelity of genetic information is essential for cellular function and viability. DNA double-strand breaks (DSBs) pose a significant threat to genome integrity, necessitating efficient repair ...mechanisms. While the predominant repair strategies are usually accurate, paradoxically, error-prone pathways also exist. This review explores recent advances and our understanding of microhomology-mediated end joining (MMEJ), an intrinsically mutagenic DSB repair pathway conserved across organisms. Central to MMEJ is the activity of DNA polymerase theta (Polθ), a specialized polymerase that fuels MMEJ mutagenicity. We examine the molecular intricacies underlying MMEJ activity and discuss its function during mitosis, where the activity of Polθ emerges as a last-ditch effort to resolve persistent DSBs, especially when homologous recombination is compromised. We explore the promising therapeutic applications of targeting Polθ in cancer treatment and genome editing. Lastly, we discuss the evolutionary consequences of MMEJ, highlighting its delicate balance between protecting genome integrity and driving genomic diversity.
Mammalian cells have evolved multiple pathways to repair DNA double strand breaks (DSBs) and ensure genome stability. In addition to non-homologous end-joining (NHEJ) and homologous recombination ...(HR), cells evolved an error-prone repair pathway termed microhomology-mediated end joining (MMEJ). The mutagenic outcome of MMEJ derives from the activity of DNA polymerase theta (Polθ) – a multidomain enzyme that is minimally expressed in normal tissue but overexpressed in tumors. Polθ expression is particularly crucial for the proliferation of HR deficient cancer cells. As a result, this mutagenic repair emerged as an attractive target for cancer therapy, and inhibitors are currently in pre-clinical development. Here, we review the multifunctionality of this enigmatic polymerase, focusing on its role during DSB repair in mammalian cells and its impact on cancer genomes.
The alternative non-homologous end-joining (NHEJ) machinery facilitates several genomic rearrangements, some of which can lead to cellular transformation. This error-prone repair pathway is triggered ...upon telomere de-protection to promote the formation of deleterious chromosome end-to-end fusions. Using next-generation sequencing technology, here we show that repair by alternative NHEJ yields non-TTAGGG nucleotide insertions at fusion breakpoints of dysfunctional telomeres. Investigating the enzymatic activity responsible for the random insertions enabled us to identify polymerase theta (Polθ; encoded by Polq in mice) as a crucial alternative NHEJ factor in mammalian cells. Polq inhibition suppresses alternative NHEJ at dysfunctional telomeres, and hinders chromosomal translocations at non-telomeric loci. In addition, we found that loss of Polq in mice results in increased rates of homology-directed repair, evident by recombination of dysfunctional telomeres and accumulation of RAD51 at double-stranded breaks. Lastly, we show that depletion of Polθ has a synergistic effect on cell survival in the absence of BRCA genes, suggesting that the inhibition of this mutagenic polymerase represents a valid therapeutic avenue for tumours carrying mutations in homology-directed repair genes.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Telomeres are distinctive structures that protect the ends of linear chromosomes and ensure genome stability. They are composed of long tracks of repetitive and G-rich DNA that is bound by shelterin, ...a dedicated six-subunit protein complex. In somatic cells, shelterin protects telomeres from the DNA damage response and regulates telomere length. Telomere repeats are replenished by telomerase, a specialized ribonucleoprotein composed of telomerase reverse transcriptase and an integral RNA component. Telomere protection and telomerase regulation have been primarily studied in somatic cells. However, recent evidence points out striking differences in the context of embryonic stem cells (ESCs). In this review, we discuss insights into telomere protection in ESCs versus somatic cells and summarize findings on telomerase regulation as a function of pluripotency.
TRF2 is dispensable for telomere protection in pluripotent cells.Loss of TRF2 in embryonic stem cells triggers a 2 cell-like stage.Alternative splicing promotes human telomerase reverse transcriptase (hTERT) mRNA accumulation in embryonic stem cells.Minor impact of hTERT transcriptional regulation as a function of pluripotency.
The relationship between gap junctional intercellular communication (GJIC) and mammary cell (CID-9) differentiation in vitro was explored. CID-9 cells differentiate and express beta-casein in an ...extracellular matrix (ECM)- and hormone-dependent manner. In response to interaction with the ECM, cells in culture modulated the expression of their gap junction proteins at the transcriptional and post-translational levels. In the presence of EHS-matrix, connexins (Cx)26, 32 and 43 localized predominantly to the plasma membrane, and enhanced GJIC as measured by Lucifer Yellow (LY) dye transfer assays was noted. Inhibition of GJIC of cells on EHS-matrix with 18 alpha glycyrrhetinic acid (GA) resulted in reversible downregulation of beta-casein expression. In the presence of cAMP, cells cultured on plastic expressed beta-casein, upregulated Cx43 and Cx26 protein levels and enhanced GJIC. This was reversed in the presence of 18 alpha GA. cAMP-treated cells plated either on a non-adhesive PolyHEMA substratum or on plastic supplemented with function-blocking anti-beta 1 integrin antibodies, maintained beta-casein expression. These studies suggest that cell-ECM interaction alone may induce differentiation through changes in cAMP levels and formation of functional gap junctions. That these events are downstream of ECM signalling was underscored by the fact that enhanced GJIC induced partial differentiation in mammary epithelial cells in the absence of an exogenously provided basement membrane and in a beta 1-integrin- and adhesion-independent manner.
Single molecule analysis of replicating DNA (SMARD) is a powerful methodology that allows in vivo analysis of replicating DNA; identification of origins of replication, assessment of fork ...directionality, and measurement of replication fork speed. SMARD, which has been extensively used to study replication of nuclear DNA, involves incorporation of thymidine analogs to nascent DNA chains and their subsequent visualization through immune detection. Here, we adapt and fine-tune the SMARD technique to the specifics of human and mouse mitochondrial DNA. The mito-SMARD protocol allows researchers to gain in vivo insight into mitochondrial DNA (mtDNA) replication at the single molecule level and with high resolution.
RHINO directs MMEJ to repair DNA breaks in mitosis Brambati, Alessandra; Sacco, Olivia; Porcella, Sarina ...
Science (American Association for the Advancement of Science),
08/2023, Letnik:
381, Številka:
6658
Journal Article
Recenzirano
Odprti dostop
Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining ...(MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9-based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes
and
and
and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.
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