The aflatoxin B-induced formamidopyrimidine adduct is repaired by transcription-coupled nucleotide excision repair in human cells.

The mycotoxin, aflatoxin B (AFB), is a potent mutagen that contaminates agricultural food supplies. After ingestion, AFB is oxidized into a reactive electrophile that alkylates DNA, forming bulky lesions such as the genotoxic formamidopyrimidine lesion, AFB-Fapy dG. This lesion is mainly repaired by nucleotide excision repair (NER) in bacteria; however, in humans the picture is less clear.

MSIanalyzer: Targeted Nanopore Sequencing Enables Single Nucleotide Resolution Analysis of Microsatellite Instability Diversity.

We present a targeted sequencing-based pipeline that profiles microsatellite instability (MSI) at single-nucleotide resolution. Targeted amplicons from the five widely studied Bethesda panel microsatellite loci were sequenced using Oxford Nanopore Technology in two microsatellite unstable colorectal cancer cell lines (HCT15, HCT116), two microsatellite stable cancer cell lines (TK6, U2OS), and two peripheral blood mononuclear cell samples from healthy donors. An anchor-extension algorithm was developed to capture repeat motifs while allowing interruptions, using a threshold informed by platform-specific error.

Comprehensive Measurement of Inter-Individual Variation in DNA Repair Capacity in Healthy Individuals.

Rare genetic DNA repair deficiency syndromes can lead to immunodeficiency, neurological disorders, and cancer. In the general population, inter-individual variation in DNA repair capacity (DRC) influences susceptibility to cancer and several age-related diseases. Genome wide association studies and functional analyses show that defects in multiple DNA repair pathways jointly increase disease risk, but previous technologies did not permit comprehensive analyses of DNA repair in populations.

Double strand breaks drive toxicity in Huntington's disease mice with or without somatic expansion.

There has been a substantial investment in elucidating the mechanism of expansion in hopes of identifying therapeutic targets for Huntington disease (HD). Although an expanded CAG allele is the causal mutation for HD, there is evidence that somatic expansion may not be the only disease driver. We report here that double strand breaks (DSBs) drive HD toxicity by an independent mechanism from somatic expansion.

Identifying active and inhibitor-resistant MGMT variants for gene therapy.

O6-methylguanine-DNA methyltransferase (MGMT) reverses alkylating-agent-induced methylation by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) at the O position of guanine. MGMT is irreversibly inhibited by O-benzylguanine (O6BG), while the Pro140Lys (P140K) variant is resistant. Combining the use of O6BG/BCNU with gene transfer of MGMT P140K into hematopoietic stem cells (HSCs) has enabled in vivo enrichment of gene-modified HSCs for therapeutic effect in preclinical studies.

Suppressed DNA repair capacity in flight attendants after air travel.

Elevated cancer risk and compromised reproductive health have been well documented in flight attendants (FA), but the etiology remains unknown. Many studies using cell and animal models suggest that air travel related exposures might plausibly explain the adverse health outcomes observed in flight crew, but our understanding of the underlying biological mechanisms is incomplete. During air travel, FA are constantly exposed to complex mixtures of mutagens in the flight cabin that may contribute to genomic instability by inducing DNA damage and interfering with DNA repair.

CHD6 has poly(ADP-ribose)- and DNA-binding domains and regulates PARP1/2-trapping inhibitor sensitivity via abasic site repair.

To tolerate oxidative stress, cells enable DNA repair responses often sensitive to poly(ADP-ribose) (PAR) polymerase 1 and 2 (PARP1/2) inhibition-an intervention effective against cancers lacking BRCA1/2. Here, we demonstrate that mutating the CHD6 chromatin remodeler sensitizes cells to PARP1/2 inhibitors in a manner distinct from BRCA1, and that CHD6 recruitment to DNA damage requires cooperation between PAR- and DNA-binding domains essential for nucleosome sliding activity. CHD6 displays direct PAR-binding, interacts with PARP-1 and other PAR-associated proteins, and combined DNA- and PAR-binding loss eliminates CHD6 relocalization to DNA damage.

DNA-PK Inhibition Shows Differential Radiosensitization in Orthotopic GBM PDX Models Based on DDR Pathway Deficits.

Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity.

Base excision repair and double strand break repair cooperate to modulate the formation of unrepaired double strand breaks in mouse brain.

We lack the fundamental information needed to understand how DNA damage in the brain is generated and how it is controlled over a lifetime in the absence of replication check points. To address these questions, here, we integrate cell-type and region-specific features of DNA repair activity in the normal brain. The brain has the same repair proteins as other tissues, but normal, canonical repair activity is unequal and is characterized by high base excision repair (BER) and low double strand break repair (DSBR).

Cosmic Ionizing Radiation: A DNA Damaging Agent That May Underly Excess Cancer in Flight Crews.

In the United States, the Federal Aviation Administration has officially classified flight crews (FC) consisting of commercial pilots, cabin crew, or flight attendants as "radiation workers" since 1994 due to the potential for cosmic ionizing radiation (CIR) exposure at cruising altitudes originating from solar activity and galactic sources. Several epidemiological studies have documented elevated incidence and mortality for several cancers in FC, but it has not yet been possible to establish whether this is attributable to CIR. CIR and its constituents are known to cause a myriad of DNA lesions, which can lead to carcinogenesis unless DNA repair mechanisms remove them.

Molecular basis and functional consequences of the interaction between the base excision repair DNA glycosylase NEIL1 and RPA.

NEIL1 is a DNA glycosylase that recognizes and initiates base excision repair of oxidized bases. The ubiquitous ssDNA binding scaffolding protein, replication protein A (RPA), modulates NEIL1 activity in a manner that depends on DNA structure. Interaction between NEIL1 and RPA has been reported, but the molecular basis of this interaction has yet to be investigated.

ATM inhibition exploits checkpoint defects and ATM-dependent double strand break repair in TP53-mutant glioblastoma.

Determining the balance between DNA double strand break repair (DSBR) pathways is essential for understanding treatment response in cancer. We report a method for simultaneously measuring non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Using this method, we show that patient-derived glioblastoma (GBM) samples with acquired temozolomide (TMZ) resistance display elevated HR and MMEJ activity, suggesting that these pathways contribute to treatment resistance.

CDK-independent role of D-type cyclins in regulating DNA mismatch repair.

Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner.

Aberrant ATM signaling and homology-directed DNA repair as a vulnerability of p53-mutant GBM to AZD1390-mediated radiosensitization.

ATM is a key mediator of radiation response, and pharmacological inhibition of ATM is a rational strategy to radiosensitize tumors. AZD1390 is a brain-penetrant ATM inhibitor and a potent radiosensitizer. This study evaluated the spectrum of radiosensitizing effects and the impact of mutation status in a panel of wild-type (WT) glioblastoma (GBM) patient-derived xenografts (PDXs).

Annual space weather fluctuations and telomere length dynamics in a longitudinal cohort of older men: the Normative Aging Study.

Background: Space weather has been associated with increased risk of cardiovascular diseases in space and flight crew. However, limited research has focused on the ground population, particularly among the elderly who are vulnerable to aging-related diseases.

Objective: We evaluated the association between space weather alterations and biological aging using leukocyte telomere length as a biomarker in healthy elderly men.

REV7 Monomer Is Unable to Participate in Double Strand Break Repair and Translesion Synthesis but Suppresses Mitotic Errors.

Rev7 is a regulatory protein with roles in translesion synthesis (TLS), double strand break (DSB) repair, replication fork protection, and cell cycle regulation. Rev7 forms a homodimer in vitro using its HORMA (Hop, Rev7, Mad2) domain; however, the functional importance of Rev7 dimerization has been incompletely understood. We analyzed the functional properties of cells expressing either wild-type mouse Rev7 or Rev7, a mutant that cannot dimerize.

Tuning ultrasmall theranostic nanoparticles for MRI contrast and radiation dose amplification.

The introduction of magnetic resonance (MR)-guided radiation treatment planning has opened a new space for theranostic nanoparticles to reduce acute toxicity while improving local control. In this work, second-generation AGuIX nanoparticles (AGuIX-Bi) are synthesized and validated. AGuIX-Bi are shown to maintain MR positive contrast while further amplifying the radiation dose by the replacement of some Gd cations with higher Z Bi.

ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs.

ATM phosphorylates the FATC domain of DNA-PK at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double strand breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PK ), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs.

Human Variation in DNA Repair, Immune Function, and Cancer Risk.

DNA damage constantly threatens genome integrity, and DNA repair deficiency is associated with increased cancer risk. An intuitive and widely accepted explanation for this relationship is that unrepaired DNA damage leads to carcinogenesis due to the accumulation of mutations in somatic cells. But DNA repair also plays key roles in the function of immune cells, and immunodeficiency is an important risk factor for many cancers.

A Human MSH6 Germline Variant Associated With Systemic Lupus Erythematosus Induces Lupus-like Disease in Mice.

Objective: To determine if single-nucleotide polymorphisms (SNPs) in DNA repair genes are enriched in individuals with systemic lupus erythematosus (SLE) and if they are sufficient to confer a disease phenotype in a mouse model.

Methods: Human exome chip data of 2499 patients with SLE and 1230 healthy controls were analyzed to determine if variants in 10 different mismatch repair genes (MSH4, EXO1, MSH2, MSH6, MLH1, MSH3, POLH, PMS2, ML3, and APEX2) were enriched in individuals with SLE. A mouse model of the MSH6 SNP, which was found to be enriched in individuals with SLE, was created using CRISPR/Cas9 gene targeting.

An Active Learning Framework Improves Tumor Variant Interpretation.

A novel machine learning approach predicts the impact of tumor mutations on cellular phenotypes, overcomes limited training data, minimizes costly functional validation, and advances efforts to implement cancer precision medicine.