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RNA–DNA hybrid is a part of the R‐loop which is an important non‐standard nucleic acid structure. RNA–DNA hybrid/R‐loop causes genomic instability by inducing DNA damages or inhibiting DNA replication. It also plays biologically important roles in regulation of transcription, replication, recombination and repair. Here, we have employed catalytically inactive human RNase H1 mutant (D145N) to visualize RNA–DNA hybrids and map their genomic locations in fission yeast cells. The RNA–DNA hybrids appear as multiple nuclear foci in rnh1∆rnh201∆ cells lacking cellular RNase H activity, but not in the wild‐type. The majority of RNA–DNA hybrid loci are detected at the protein coding regions and tRNA. In rnh1∆rnh201∆ cells, cells with multiple Rad52 foci increase during S‐phase and about 20% of the RNA–DNA hybrids overlap with Rad52 loci. During S‐phase, more robust association of Rad52 with RNA–DNA hybrids was observed in the protein coding region than in M‐phase. These results suggest that persistent RNA–DNA hybrids in the protein coding region in rnh1∆rnh201∆ cells generate DNA damages during S‐phase, potentially through collision with DNA replication forks. [ABSTRACT FROM AUTHOR]

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1−/− and TDP2−/− cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1−/− and RAD54−/− suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde. [ABSTRACT FROM AUTHOR]

Steroid hormones induce the transcription of target genes by activating nuclear receptors. Early transcriptional response to various stimuli, including hormones, involves the active catalysis of topoisomerase II (TOP2) at transcription regulatory sequences. TOP2 untangles DNAs by transiently generating double‐strand breaks (DSBs), where TOP2 covalently binds to DSB ends. When TOP2 fails to rejoin, called "abortive" catalysis, the resulting DSBs are repaired by tyrosyl‐DNA phosphodiesterase 2 (TDP2) and non‐homologous end‐joining (NHEJ). A steroid, cortisol, is the most important glucocorticoid, and dexamethasone (Dex), a synthetic glucocorticoid, is widely used for suppressing inflammation in clinics. We here revealed that clinically relevant concentrations of Dex and physiological concentrations of cortisol efficiently induce DSBs in G1 phase cells deficient in TDP2 and NHEJ. The DSB induction depends on glucocorticoid receptor (GR) and TOP2. Considering the specific role of TDP2 in removing TOP2 adducts from DSB ends, induced DSBs most likely represent stalled TOP2‐DSB complexes. Inhibition of RNA polymerase II suppressed the DSBs formation only modestly in the G1 phase. We propose that cortisol and Dex frequently generate DSBs through the abortive catalysis of TOP2 at transcriptional regulatory sequences, including promoters or enhancers, where active TOP2 catalysis occurs during early transcriptional response. [ABSTRACT FROM AUTHOR]

Base excision repair (BER) removes damaged bases by generating single‐strand breaks (SSBs), gap‐filling by DNA polymerase β (POLβ), and resealing SSBs. A base‐damaging agent, methyl methanesulfonate (MMS) is widely used to study BER. BER increases cellular tolerance to MMS, anti‐cancer base‐damaging drugs, temozolomide, carmustine, and lomustine, and to clinical poly(ADP ribose)polymerase (PARP) poisons, olaparib and talazoparib. The poisons stabilize PARP1/SSB complexes, inhibiting access of BER factors to SSBs. PARP1 and XRCC1 collaboratively promote SSB resealing by recruiting POLβ to SSBs, but XRCC1−/− cells are much more sensitive to MMS than PARP1−/− cells. We recently report that the PARP1 loss in XRCC1−/− cells restores their MMS tolerance and conclude that XPCC1 facilitates the release of PARP1 from SSBs by maintaining its autoPARylation. We here show that the PARP1 loss in XRCC1−/− cells also restores their tolerance to the three anti‐cancer base‐damaging drugs, although they and MMS induce different sets of base damage. We reveal the synthetic lethality of the XRCC1−/− mutation, but not POLβ−/−, with olaparib and talazoparib, indicating that XRCC1 is a unique BER factor in suppressing toxic PARP1/SSB complex and can suppress even when PARP1 catalysis is inhibited. In conclusion, XRCC1 suppresses the PARP1/SSB complex via PARP1 catalysis‐dependent and independent mechanisms. [ABSTRACT FROM AUTHOR]