Ku limits RNA-induced innate immunity to allow Alu expansion in primates.

Ku70 and Ku80 form the Ku heterodimer, a ring-shaped complex that initiates the non-homologous end-joining (NHEJ) DNA repair pathway. Ku binds to double-stranded DNA ends and recruits other NHEJ factors, including LIG4 and DNA-PKcs. Although Ku can bind to double-stranded RNA (dsRNA) and trap mutated DNA-PKcs on ribosomal RNA, the physiological role of the Ku-RNA interaction in otherwise wild-type cells remains unclear.

Ku suppresses RNA-mediated innate immune responses in human cells to accommodate primate-specific Alu expansion.

Ku70 and Ku80 form Ku, a ring-shaped protein that initiates the non-homologous end-joining (NHEJ) DNA repair pathway. Specifically, Ku binds to double-stranded DNA (dsDNA) ends and recruits other NHEJ factors ( , DNA-PKcs and LIG4). While Ku binds to double-stranded RNA (dsRNA) and traps mutated-DNA-PKcs on ribosomal RNA the physiological significance of Ku-dsRNA interactions in otherwise wild-type cells remains elusive.

Lig3-dependent rescue of mouse viability and DNA double-strand break repair by catalytically inactive Lig4.

Recent studies have revealed a structural role for DNA ligase 4 (Lig4) in the maintenance of a repair complex during non-homologous end joining (NHEJ) of DNA double-strand breaks. In cultured cell lines, catalytically inactive Lig4 can partially alleviate the severe DNA repair phenotypes observed in cells lacking Lig4. To study the structural role of Lig4 in vivo, a mouse strain harboring a point mutation to Lig4's catalytic site was generated.

DNA repair and anti-cancer mechanisms in the long-lived bowhead whale.

At over 200 years, the maximum lifespan of the bowhead whale exceeds that of all other mammals. The bowhead is also the second-largest animal on Earth, reaching over 80,000 kg. Despite its very large number of cells and long lifespan, the bowhead is not highly cancer-prone, an incongruity termed Peto's Paradox.

The KU70-SAP domain has an overlapping function with DNA-PKcs in limiting the lateral movement of KU along DNA.

Unlabelled: The non-homologous end-joining (NHEJ) pathway is critical for DNA double-strand break repair and is essential for lymphocyte development and maturation. The Ku70/Ku80 heterodimer (KU) binds to DNA ends, initiating NHEJ and recruiting additional factors, including DNA-dependent protein kinase catalytic subunit (DNA-PKcs) that caps the ends and pushes KU inward. The C-terminus of Ku70 in higher eukaryotes includes a flexible linker and a SAP domain, whose physiological role remains poorly understood.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates in vitro.

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70-Ku80 heterodimer (Ku), X-ray repair cross complementing 4 (XRCC4) in complex with DNA ligase 4 (X4L4) and XRCC4-like factor (XLF) form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) were recently obtained by single-particle cryo-electron microscopy analysis.

Targeting NUPR1-dependent stress granules formation to induce synthetic lethality in Kras-driven tumors.

We find that NUPR1, a stress-associated intrinsically disordered protein, induced droplet formation via liquid-liquid phase separation (LLPS). NUPR1-driven LLPS was crucial for the creation of NUPR1-dependent stress granules (SGs) in pancreatic cancer cells since genetic or pharmacological inhibition by ZZW-115 of NUPR1 activity impeded SGs formation. The Kras mutation induced oncogenic stress, NUPR1 overexpression, and promoted SGs development.

Unravelling the complexities of DNA-PK activation by structure-based mutagenesis.

It has been known for decades that the DNA-dependent protein kinase (DNA-PK) is only an active serine/threonine protein kinase when it is bound to a DNA double-stranded end; still, the molecular details of how this activation is achieved have remained elusive. The recent surge in structural information for DNA-PK complexes has provided valuable insights into the process of DNA end recognition by DNA-PK. A particularly intriguing feature of this kinase is a region of the protein that can transition from a seemingly structurally disordered state to a single alpha-helix that traverses down the DNA binding cradle.

CRISPR-dependent Base Editing Screens Identify Separation of Function Mutants of RADX with Altered RAD51 Regulatory Activity.

RAD51 forms nucleoprotein filaments to promote homologous recombination, replication fork reversal, and fork protection. Numerous factors regulate the stability of these filaments and improper regulation leads to genomic instability and ultimately disease including cancer. RADX is a single stranded DNA binding protein that modulates RAD51 filament stability.

Multivalent interactions of the disordered regions of XLF and XRCC4 foster robust cellular NHEJ and drive the formation of ligation-boosting condensates .

In mammalian cells, DNA double-strand breaks are predominantly repaired by non-homologous end joining (NHEJ). During repair, the Ku70/80 heterodimer (Ku), XRCC4 in complex with DNA Ligase 4 (X4L4), and XLF form a flexible scaffold that holds the broken DNA ends together. Insights into the architectural organization of the NHEJ scaffold and its regulation by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) have recently been obtained by single-particle cryo-electron microscopy analysis.

The SMC-like RecN protein is at the crossroads of several genotoxic stress responses in .

Introduction: DNA damage repair (DDR) is an essential process for living organisms and contributes to genome maintenance and evolution. DDR involves different pathways including Homologous recombination (HR), Nucleotide Excision Repair (NER) and Base excision repair (BER) for example. The activity of each pathway is revealed with particular drug inducing lesions, but the repair of most DNA lesions depends on concomitant or subsequent action of the multiple pathways.

RAD51AP1 regulates ALT-HDR through chromatin-directed homeostasis of TERRA.

Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in subsets of aggressive cancer. Recent studies have revealed that telomere repeat-containing RNA (TERRA) promotes ALT-associated HDR (ALT-HDR). Here, we report that RAD51AP1, a crucial ALT factor, interacts with TERRA and utilizes it to generate D- and R-loop HR intermediates.

Catalytically inactive DNA ligase IV promotes DNA repair in living cells.

DNA double strand breaks (DSBs) are induced by external genotoxic agents (ionizing radiation or genotoxins) or by internal processes (recombination intermediates in lymphocytes or by replication errors). The DNA ends induced by these genotoxic processes are often not ligatable, requiring potentially mutagenic end-processing to render ends compatible for ligation by non-homologous end-joining (NHEJ). Using single molecule approaches, Loparo et al.

Homologous recombination-deficient mutation cluster in tumor suppressor identified by comprehensive analysis of cancer variants.

Mutations in homologous recombination (HR) genes, including , , and the RAD51 paralog , predispose to tumorigenesis and sensitize cancers to DNA-damaging agents and poly(ADP ribose) polymerase inhibitors. However, ∼800 missense variants of unknown significance have been identified for RAD51C alone, impairing cancer risk assessment and therapeutic strategies. Here, we interrogated >50 RAD51C missense variants, finding that mutations in residues conserved with RAD51 strongly predicted HR deficiency and disrupted interactions with other RAD51 paralogs.

Simultaneous Mechanical and Fluorescence Detection of Helicase-Catalyzed DNA Unwinding.

Helicases are ubiquitous molecular motor proteins that utilize the energy derived from the hydrolysis of nucleoside triphosphates (NTPs) to transiently convert the duplex form of nucleic acids to single-stranded intermediates for many biological processes. These enzymes play vital roles in nearly all aspects of nucleic acid metabolism, such as DNA repair and RNA splicing. Understanding helicase's functional roles requires methods to dissect the mechanisms of motor proteins at the molecular level.

Bloom Syndrome Helicase Compresses Single-Stranded DNA into Phase-Separated Condensates.

Bloom syndrome protein (BLM) is a conserved RecQ family helicase involved in the maintenance of genome stability. BLM has been widely recognized as a genome "caretaker" that processes structured DNA. In contrast, our knowledge of how BLM behaves on single-stranded (ss) DNA is still limited.

The convergence of head-on DNA unwinding forks induces helicase oligomerization and activity transition.

Helicases are multifunctional motor proteins with the primary task of separating nucleic acid duplexes. These enzymes often exist in distinct oligomeric forms and play essential roles during nucleic acid metabolism. Whether there is a correlation between their oligomeric state and cellular function, and how helicases effectively perform functional switching remains enigmatic.

STING protects breast cancer cells from intrinsic and genotoxic-induced DNA instability via a non-canonical, cell-autonomous pathway.

STING (Stimulator of Interferon Genes) is an endoplasmic reticulum-anchored adaptor of the innate immunity best known to trigger pro-inflammatory cytokine expression in response to pathogen infection. In cancer, this canonical pathway can be activated by intrinsic or drug-induced genomic instability, potentiating antitumor immune responses. Here we report that STING downregulation decreases cell survival and increases sensitivity to genotoxic treatment in a panel of breast cancer cell lines in a cell-autonomous manner.

Insights into the control of RAD51 nucleoprotein filament dynamics from single-molecule studies.

Genomic integrity depends on the RecA/RAD51 protein family. Discovered over five decades ago with the founder bacterial RecA protein, eukaryotic RAD51 is an ATP-dependent DNA strand transferase implicated in DNA double-strand break and single-strand gap repair, and in dealing with stressed DNA replication forks. RAD51 assembles as a nucleoprotein filament around single-stranded DNA to promote homology recognition in a duplex DNA and subsequent strand exchange.

A basal-level activity of ATR links replication fork surveillance and stress response.

Mammalian cells use diverse pathways to prevent deleterious consequences during DNA replication, yet the mechanism by which cells survey individual replisomes to detect spontaneous replication impediments at the basal level, and their accumulation during replication stress, remain undefined. Here, we used single-molecule localization microscopy coupled with high-order-correlation image-mining algorithms to quantify the composition of individual replisomes in single cells during unperturbed replication and under replicative stress. We identified a basal-level activity of ATR that monitors and regulates the amounts of RPA at forks during normal replication.

Distinct pathways of homologous recombination controlled by the SWS1-SWSAP1-SPIDR complex.

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR.

Single-molecule imaging reveals replication fork coupled formation of G-quadruplex structures hinders local replication stress signaling.

Guanine-rich DNA sequences occur throughout the human genome and can transiently form G-quadruplex (G4) structures that may obstruct DNA replication, leading to genomic instability. Here, we apply multi-color single-molecule localization microscopy (SMLM) coupled with robust data-mining algorithms to quantitatively visualize replication fork (RF)-coupled formation and spatial-association of endogenous G4s. Using this data, we investigate the effects of G4s on replisome dynamics and organization.

Dynamics of Ku and bacterial non-homologous end-joining characterized using single DNA molecule analysis.

We use single-molecule techniques to characterize the dynamics of prokaryotic DNA repair by non-homologous end-joining (NHEJ), a system comprised only of the dimeric Ku and Ligase D (LigD). The Ku homodimer alone forms a ∼2 s synapsis between blunt DNA ends that is increased to ∼18 s upon addition of LigD, in a manner dependent on the C-terminal arms of Ku. The synapsis lifetime increases drastically for 4 nt complementary DNA overhangs, independently of the C-terminal arms of Ku.

RADX controls RAD51 filament dynamics to regulate replication fork stability.

The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication.

A Disease-Causing Single Amino Acid Deletion in the Coiled-Coil Domain of RAD50 Impairs MRE11 Complex Functions in Yeast and Humans.

The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here, we identify a patient with bone marrow failure and developmental defects caused by biallelic RAD50 mutations. One of the mutations creates a null allele, whereas the other (RAD50) leads to the loss of a single residue in the heptad repeats within the RAD50 coiled-coil domain.