Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.

Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

Recent whole-exome sequencing of malignancies have detected recurrent somatic mutations in U2 small nuclear ribonucleoprotein complex (snRNP) components of the spliceosome. These factors have also been identified as novel players in the DNA-damage response (DDR) in several genome-wide screens and proteomic analysis. Although accumulating evidence implies that the spliceosome has an important role in genome stability and is an emerging hallmark of cancer, its precise role in DNA repair still remains elusive. Here we identify two distinct mechanisms of how spliceosome U2 snRNP factors contribute to genome stability. We show that the spliceosome maintains protein levels of essential repair factors, thus contributing to homologous recombination repair. In addition, real-time laser microirradiation analysis identified rapid recruitment of the U2 snRNP factor SNRPA1 to DNA-damage sites. Functional analysis of SNRPA1 revealed a more immediate and direct role in preventing R-loop-induced DNA damage. Our present study implies a complex interrelation between transcription, mRNA splicing and the DDR. Cells require rapid spatio-temporal coordination of these chromatin transactions to cope with various forms of genotoxic stress.

Evidence that long non-coding RNAs (lncRNAs) participate in DNA repair is accumulating, however, whether they can control DNA repair pathway choice is unknown. Here we show that the small Cajal body-specific RNA 2 (scaRNA2) can promote HR by inhibiting DNA-dependent protein kinase (DNA-PK) and, thereby, NHEJ. By binding to the catalytic subunit of DNA-PK (DNA-PKcs), scaRNA2 weakens its interaction with the Ku70/80 subunits, as well as with the LINP1 lncRNA, thereby preventing catalytic activation of the enzyme. Inhibition of DNA-PK by scaRNA2 stimulates DNA end resection by the MRN/CtIP complex, activation of ATM at DNA lesions and subsequent repair by HR. ScaRNA2 is regulated in turn by WRAP53β, which binds this RNA, sequestering it away from DNA-PKcs and allowing NHEJ to proceed. These findings reveal that RNA-dependent control of DNA-PK catalytic activity is involved in regulating whether the cell utilizes NHEJ or HR.

8-oxoguanine DNA glycosylase (OGG1) is the main DNA glycosylase responsible for the excision of 7,8-dihydro-8-oxoguanine (8-oxoG) from duplex DNA to initiate base excision repair. This glycosylase activity is relevant in many pathological conditions including cancer, inflammation, and neurodegenerative diseases. To have a better understanding of the role of OGG1, we previously reported TH5487, a potent active site inhibitor of OGG1. Here, we further investigate the consequences of inhibiting OGG1 with TH5487. TH5487 treatment induces accumulation of genomic 8-oxoG lesions. Furthermore, it impairs the chromatin binding of OGG1 and results in lower recruitment of OGG1 to regions of DNA damage. Inhibiting OGG1 with TH5487 interferes with OGG1′s incision activity, resulting in fewer DNA double-strand breaks in cells exposed to oxidative stress. This study validates TH5487 as a potent OGG1 inhibitor that prevents the repair of 8-oxoG and alters OGG1–chromatin dynamics and OGG1′s recruitment kinetics.