Phase separation forms membraneless compartments in the nuclei, including by establishing heterochromatin \"domains\" and repair foci. Pericentromeric heterochromatin mostly comprises repeated sequences prone to aberrant recombination, and \"safe\" homologous recombination (HR) repair of these sequences requires the movement of repair sites to the nuclear periphery before Rad51 recruitment and strand invasion. How this mobilization initiates is unknown, and the contribution of phase separation to these dynamics is unclear. Here, we show that Nup98 nucleoporin is recruited to heterochromatic repair sites before relocalization through Sec13 or Nup88 nucleoporins, and downstream from the Smc5/6 complex and SUMOylation. Remarkably, the phase separation properties of Nup98 are required and sufficient to mobilize repair sites and exclude Rad51, thus preventing aberrant recombination while promoting HR repair. Disrupting this pathway results in heterochromatin repair defects and widespread chromosome rearrangements, revealing a novel \"off-pore\" role for nucleoporins and phase separation in nuclear dynamics and genome integrity in a multicellular eukaryote.

DNA double-strand break (DSB) repair is a fundamental cellular process crucial for maintaining genome stability, with homologous recombination and non-homologous end joining as the primary mechanisms, and various alternative pathways such as single-strand annealing (SSA) and microhomology-mediated end joining also playing significant roles under specific conditions. IRC genes were previously identified as part of a group of genes associated with increased levels of Rad52 foci in Saccharomyces cerevisiae. In this study, we investigated the effects of IRC gene mutations on DSB repair, focusing on uncharacterized IRC10, 19, 21, 22, 23, and 24. Gene conversion (GC) assay revealed that irc10Δ, 22Δ, 23Δ, and 24Δ mutants displayed modest increases in GC frequencies, while irc19Δ and irc21Δ mutants exhibited significant reductions. Further investigation revealed that deletion mutations in URA3 were not generated in irc19Δ mutant cells following HO-induced DSBs. Additionally, irc19Δ significantly reduced frequency of SSA, and a synergistic interaction between irc19Δ and rad52Δ was observed in DSB repair via SSA. Assays to determine the choice of DSB repair pathways indicated that Irc19 is necessary for generating both GC and deletion products. Overall, these results suggest a potential role of Irc19 in DSB repair pathways, particularly in end resection process.

DNA double-strand breaks (DSBs) elicit an elaborate response to signal damage and trigger repair via two major pathways: nonhomologous end-joining (NHEJ), which functions throughout the interphase, and homologous recombination (HR), restricted to S/G2 phases. The DNA damage response relies, on post-translational modifications of nuclear factors to coordinate the mending of breaks. Ubiquitylation of histones and chromatin-associated factors regulates DSB repair and numerous E3 ubiquitin ligases are involved in this process. Despite significant progress, our understanding of ubiquitin-mediated DNA damage response regulation remains incomplete. Here, we have performed a localization screen to identify RING/U-box E3 ligases involved in genome maintenance. Our approach uncovered 7 novel E3 ligases that are recruited to microirradiation stripes, suggesting potential roles in DNA damage signaling and repair. Among these factors, the DELTEX family E3 ligase DTX2 is rapidly mobilized to lesions in a poly ADP-ribosylation-dependent manner. DTX2 is recruited and retained at DSBs via its WWE and DELTEX conserved C-terminal domains. In cells, both domains are required for optimal binding to mono and poly ADP-ribosylated proteins with WWEs playing a prominent role in this process. Supporting its involvement in DSB repair, DTX2 depletion decreases HR efficiency and moderately enhances NHEJ. Furthermore, DTX2 depletion impeded BRCA1 foci formation and increased 53BP1 accumulation at DSBs, suggesting a fine-tuning role for this E3 ligase in repair pathway choice. Finally, DTX2 depletion sensitized cancer cells to X-rays and PARP inhibition and these susceptibilities could be rescued by DTX2 reexpression. Altogether, our work identifies DTX2 as a novel ADP-ribosylation-dependent regulator of HR-mediated DSB repair.

Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks (DSBs) as cytotoxic DNA damage; thus, the DSB repair activity in each cancer cell significantly influences the efficacy of the treatments. Pancreatic cancers are known to be resistant to these treatments, and the overexpression of MUC1, a member of the glycoprotein mucins, is associated with IR- and chemo-resistance. Therefore, we investigated the impact of MUC1 on DSB repair. This report examined the effect of the overexpression of MUC1 on homologous recombination (HR) and non-homologous end-joining (NHEJ) using cell-based DSB repair assays. In addition, the therapeutic potential of NHEJ inhibitors including HDAC inhibitors was also studied using pancreatic cancer cell lines. The MUC1-overexpression enhances NHEJ, while partially suppressing HR. Also, MUC1-overexpressed cancer cell lines are preferentially killed by a DNA-PK inhibitor and HDAC1/2 inhibitors. Altogether, MUC1 induces metabolic changes that create an imbalance between NHEJ and HR activities, and this imbalance can be a target for selective killing by HDAC inhibitors. This is a novel mechanism of MUC1-mediated IR-resistance and will form the basis for targeting MUC1-overexpressed pancreatic cancer.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancer can also be driven purely by epigenetic alterations, without driver mutations. Specifically, a 24-h transient downregulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to induce epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 downregulation is performed for extended periods of time remains unclear. Here, we show that prolonged depletion of PH, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad misregulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects accumulate during the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

Non-Hispanic Black breast cancer survivors have poorer outcomes and higher mortality rates than White survivors, but systemic biological mechanisms underlying these disparities are unclear. We used circulating leukocytes as a surrogate for measuring systemic mechanisms, which might be different from processes in the target tissue (e.g., breast). We investigated race-based differences in DNA damage and repair, using a novel CometChip assay, in circulating leukocytes from breast cancer survivors who had completed primary cancer therapy and were cancer free. We observed novel race-based differences in systemic DNA damage and repair activity in cancer survivors, but not in cells from healthy volunteers. Basal DNA damage in leukocytes was higher in White survivors, but Black survivors showed a much higher induction after bleomycin treatment. Double-strand break repair activity was also significantly different between the races, with cells from White survivors showing more sustained repair activity compared to Black leukocytes. These results suggest that cancer and cancer therapy might have long-lasting effects on systemic DNA damage and repair mechanisms that differ in White survivors and Black survivors. Findings from our preliminary study in non-cancer cells (circulating leukocytes) suggest systemic effects beyond the target site, with implications for accelerated aging-related cancer survivorship disparities.

Cancer initiation and progression are typically associated with the accumulation of driver mutations and genomic instability. However, recent studies demonstrated that cancers can also be purely initiated by epigenetic alterations, without driver mutations. Specifically, a 24-hours transient down-regulation of polyhomeotic (ph-KD), a core component of the Polycomb complex PRC1, is sufficient to drive epigenetically initiated cancers (EICs) in Drosophila, which are proficient in DNA repair and are characterized by a stable genome. Whether genomic instability eventually occurs when PRC1 down-regulation is performed for extended periods of time remains unclear. Here we show that prolonged depletion of a PRC1 component, which mimics cancer initiating events, results in broad dysregulation of DNA replication and repair genes, along with the accumulation of DNA breaks, defective repair, and widespread genomic instability in the cancer tissue. A broad mis-regulation of H2AK118 ubiquitylation and to a lesser extent of H3K27 trimethylation also occurs, and might contribute to these phenotypes. Together, this study supports a model where DNA repair and replication defects amplify the tumorigenic transformation epigenetically induced by PRC1 loss, resulting in genomic instability and cancer progression.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

OBJECTIVE: Numerous studies have reported the over-expression of the radiation-sensitive protein 51 (RAD51) in various types of cancer. However, the role of RAD51 genotypes in lung cancer remains largely unknown. This study aimed to assess the impact of the common variant RAD51 rs1801320 (G-135C) genotypes on the risk of lung cancer in Taiwan.
METHODS: The contribution of RAD51 rs1801320 genotypes to lung cancer risk was investigated in a cohort comprising 358 lung cancer patients and 716 age- and sex-matched healthy controls, utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methodology.
RESULTS: The analysis revealed that among the control subjects, the percentages of GG, CG, and CC genotypes of RAD51 rs1801320 were 73.2%, 24.3%, and 2.5%, respectively. Among the lung cancer patients, these percentages were 71.0%, 25.1%, and 3.9%, respectively (p for trend=0.4075). Allelic frequency distributions showed no significant association between the C allele of RAD51 rs1801320 and lung cancer risk determination (p=0.2987). Specifically, the RAD51 rs1801320 CC genotypes were associated with an elevated risk of lung cancer among males [adjusted odds ratio (aOR)=2.28, 95% confidence interval (95%CI)=1.03-4.87] and smokers (aOR=2.93, 95%CI=1.23-5.87), but not among females and non-smokers.
CONCLUSIONS: The RAD51 rs1801320 CC genotype was identified as a risk factor for elevated lung cancer risk in males and smokers. This genotype may serve as a molecular biomarker at the DNA level for early detection and prediction of lung cancer in Taiwan.

Human Replication Protein A (RPA) was historically discovered as one of the six components needed to reconstitute simian virus 40 DNA replication from purified components. RPA is now known to be involved in all DNA metabolism pathways that involve single-stranded DNA (ssDNA). Heterotrimeric RPA comprises several domains connected by flexible linkers and is heavily regulated by post-translational modifications (PTMs). The structure of RPA has been challenging to obtain. Various structural methods have been applied, but a complete understanding of RPA\'s flexible structure, its function, and how it is regulated by PTMs has yet to be obtained. This review will summarize recent literature concerning how RPA is phosphorylated in the cell cycle, the structural analysis of RPA, DNA and protein interactions involving RPA, and how PTMs regulate RPA activity and complex formation in double-strand break repair. There are many holes in our understanding of this research area. We will conclude with perspectives for future research on how RPA PTMs control double-strand break repair in the cell cycle.