ABSTRACT: CGH of stage 13 amplifying follicle cells to measure changes in replication fork progression in DNA damage checkpoint and double-strand break repair mutants
Project description:CGH of stage 13 amplifying follicle cells to measure changes in replication fork progression in double-strand break repair mutants Comparative genomic hybridization was performed to compare amplification gradients of stage 13 follicle cells from several double-strand break repair mutants to wild type (OrR) gradients. Two-three replicates were done for each genotype.
Project description:Comparative genomic hybridization was performed to compare amplification gradients of stage 13 follicle cells from several DNA damage checkpoint and double-strand break repair mutants to wild-type (OrR) gradients. Two-three replicates were done for each genotype.
Project description:Transcription hinders replication fork progression and stability, and the Mec1/ATR checkpoint protects fork integrity. Examining checkpoint-dependent mechanisms controlling fork stability, we find that fork reversal or dormant origin firing owing to checkpoint defects are rescued in checkpoint mutants lacking THO, TREX-2 or inner basket nucleoporins. Gene gating tethers transcribed genes to the nuclear periphery and is counteracted by checkpoint kinases through phosphorylation of nucleoporins such as Mlp1. Checkpoint mutants fail to detach transcribed genes from nuclear pores, thus generating topological impediments for incoming forks. Releasing this topological complexity by introducing a double-strand break between a fork and a transcribed unit prevents fork collapse. Mlp1 mutants mimicking constitutive checkpoint-dependent phosphorylation also alleviate checkpoint defects. We propose that the checkpoint assists fork progression and stability at transcribed genes by phosphorylating key nucleoporins and counteracting gene gating, thus neutralizing the topological tension generated at nuclear pore gated genes.
Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci (RPLs) that become processed into persistent double-strand breaks across the mammalian genome from ATR inhibition.
Project description:Transient obstruction of DNA polymerase progression activates the ATR checkpoint kinase, which suppresses fork breakage, strand resection, and RPA accumulation. Herein, we use a developed DNA break-detection assay, BrITL, to identify replication-problematic loci that become processed into persistent double-strand breaks across the human genome from ATR inhibition.
Project description:Recent observations show that the single-cell response of p53 to ionizing radiation (IR) is “digital” in that it is the number of oscillations rather than the amplitude of p53 that shows dependence on the radiation dose. We present a model of this phenomenon. In our model, double-strand break (DSB) sites induced by IR interact with a limiting pool of DNA repair proteins, forming DSB–protein complexes at DNA damage foci. The persisting complexes are sensed by ataxia telangiectasia mutated (ATM), a protein kinase that activates p53 once it is phosphorylated by DNA damage. The ATM-sensing module switches on or off the downstream p53 oscillator, consisting of a feedback loop formed by p53 and its negative regulator, Mdm2. In agreement with experiments, our simulations show that by assuming stochasticity in the initial number of DSBs and the DNA repair process, p53 and Mdm2 exhibit a coordinated oscillatory dynamics upon IR stimulation in single cells, with a stochastic number of oscillations whose mean increases with IR dose. The damped oscillations previously observed in cell populations can be explained as the aggregate behavior of single cell
Project description:RAD51, a multifunctional protein, plays a central role in DNA replication and homologous recombination repair, and is known to be involved in cancer development. We identified a novel role for RAD51 in innate immune response signaling. Defects in RAD51 lead to the accumulation of self-DNA in the cytoplasm, triggering a STING-mediated innate immune response after replication stress and DNA damage. Our data suggest that in addition to playing roles in homologous recombination-mediated DNA double-strand break repair and replication fork processing, RAD51 is also implicated in the suppression of innate immunity. Thus, our study reveals a previously uncharacterized role of RAD51 in initiating immune signaling, placing it at the hub of new interconnections between DNA replication, DNA repair, and immunity.
Project description:The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among double strand break (DSB) repair proteins for its association with the DNA replication fork. Here we show that Mre11 complex inactivation causes DNA replication stress and changes in the abundance of proteins associated with nascent DNA. One of the most highly enriched proteins at the DNA replication fork upon Mre11 complex inactivation was the ubiquitin like protein ISG15. Mre11 complex deficiency and drug induced replication stress both led to the accumulation of cytoplasmic DNA and the subsequent activation of innate immune signaling via cGAS-STING-Tbk1. This led to ISG15 induction and protein ISGylation, including constituents of the replication fork. ISG15 plays a direct role in preventing replication stress. Deletion of ISG15 was associated with replication fork stalling, tonic ATR activation, genomic aberrations, and sensitivity to aphidicolin. These data reveal a previously unrecognized role for ISG15 in mitigating DNA replication stress and promoting DNA replication fidelity.
