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Humans are exposed to DNA alkylating agents through endogenous metabolism, environmental exposure and cancer chemotherapy. The resulting alkylated DNA adducts may elicit genome instability by perturbing DNA replication and transcription. R-loops regulate various cellular processes, including transcription, DNA repair, and telomere maintenance. However, unscheduled R-loops are also recognized as potential sources of DNA damage and genome instability. In this study, by employing fluorescence microscopy and R-loop sequencing approaches, we uncovered, for the first time, that minor-groove N2-alkyl-dG lesions elicit elevated R-loop accumulation in chromatin and in plasmid DNA in cells. We also demonstrated that the N2-alkyl-dG-induced R-loops impede transcription elongation and compromise genome integrity. Moreover, genetic depletion of DDX23, a R-loop helicase, renders cells more sensitive toward benzo[a]pyrene diolepoxide, a carcinogen that induces mainly the minor-groove N2-dG adduct. Together, our work unveiled that unrepaired minor-groove N2-alkyl-dG lesions may perturb genome integrity through augmenting R-loop levels in chromatin. Our findings suggest a potential therapeutic strategy involving the combination of R-loop helicase inhibitors with DNA alkylating drugs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

N 2 -Alkyl-2'-deoxyguanosine ( N 2 -alkyl-dG) is a major type of minor-groove DNA lesions arising from endogenous metabolic processes and exogenous exposure to environmental contaminants. The N 2 -alkyl-dG lesions, if left unrepaired, can block DNA replication and transcription and induce mutations in these processes. Nevertheless, the repair pathways for N 2 -alkyl-dG lesions remain incompletely elucidated. By utilizing a photo-cross-linking coupled with mass spectrometry-based quantitative proteomic analysis, we identified a series of candidate N 2 -alkyl-dG-binding proteins. We found that two of these proteins, i.e., high-mobility group protein B3 (HMGB3) and SUB1, could bind directly to N 2 - n Bu-dG-containing duplex DNA in vitro and promote the repair of this lesion in cultured human cells. In addition, HMGB3 and SUB1 protected cells against benzo[ a ]pyrene-7,8-diol-9,10-epoxide (BPDE). SUB1 exhibits preferential binding to both the cis and trans diastereomers of N 2 -BPDE-dG over unmodified dG. On the other hand, HMGB3 binds favorably to trans - N 2 -BPDE-dG; the protein, however, does not distinguish cis - N 2 -BPDE-dG from unmodified dG. Consistently, genetic ablation of HMGB3 conferred diminished repair of trans - N 2 -BPDE-dG, but not its cis counterpart, whereas loss of SUB1 conferred attenuated repair of both diastereomers. Together, we identified proteins involved in the cellular sensing and repair of minor-groove N 2 -alkyl-dG lesions and documented a unique role of HMGB3 in the stereospecific recognition and repair of N 2 -BPDE-dG.

Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N  2 -heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N  2 -modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome., Competing Interests: Competing interests statement:The authors declare no competing interest.

Dietary exposure to aflatoxin B 1 (AFB 1 ) is a recognized risk factor for developing hepatocellular carcinoma. The mutational signature of AFB 1 is characterized by high-frequency base substitutions, predominantly G>T transversions, in a limited subset of trinucleotide sequences. The 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) has been implicated as the primary DNA lesion responsible for AFB 1 -induced mutations. This study evaluated the mutagenic potential of AFB 1 -FapyGua in four sequence contexts, including hot- and cold-spot sequences as apparent in the mutational signature. Vectors containing site-specific AFB 1 -FapyGua lesions were replicated in primate cells and the products of replication were isolated and sequenced. Consistent with the role of AFB 1 -FapyGua in AFB 1 -induced mutagenesis, AFB 1 -FapyGua was highly mutagenic in all four sequence contexts, causing G>T transversions and other base substitutions at frequencies of ~80%-90%. These data suggest that the unique mutational signature of AFB 1 is not explained by sequence-dependent fidelity of replication past AFB 1 -FapyGua lesions., (© 2023 Environmental Mutagenesis and Genomics Society.)

Recognition and repair of DNA lesions are critical for cell survival. Herein, we highlight recent advances in the sequencing, repair mechanisms, and biological consequences of DNA lesions presented at the 2022 Fall American Chemical Society meeting.

O 6 -Methyl-2'-deoxyguanosine ( O 6 -MeG) is one of the most common DNA lesions and arises as a consequence of both xenobiotic carcinogens and endogenous methylation by S -adenosylmethionine. O 6 -MeG frequently causes G-to-A mutations during DNA replication due to the misincorporation of dTTP and continued DNA synthesis. Efforts to detect DNA adducts such as O 6 -MeG, and to understand their impacts on DNA structure and function, have motivated the creation of nucleoside analogs with altered base moieties to afford a more favorable interaction with the adduct as compared to the unmodified nucleotide. Such analogs directed at O 6 -MeG include benzimidazolinone and benzimidazole nucleotides, as well as their extended π surface analogs naphthimidazolinone and napthimidazole derivatives. These analogs form a more stable pair with O 6 -MeG than with G, most likely due to a combination of H-bonding and stacking. While extending the π surface of the analogs enhances their performance as adduct-directed probes, the precise origins of the increased affinity between the synthetic analogs and O 6 -MeG remain unclear. To better understand relevant conformational and pairing properties, we used X-ray crystallography and analyzed the structures of the DNA duplexes with naphthimidazolinone inserted opposite G or O 6 -MeG. The structures reveal a complex interaction of the analog found either in an anti orientation and stacked inside the duplex, either above or below G or O 6 -MeG, or in a syn orientation and paired opposite G with formation of a single H-bond. The experimental structural data are consistent with the stabilizing effect of the synthetic analog observed in UV melting experiments and calculations and moreover reveal that the origin of these observations appears to be superior stacking between O 6 -MeG and the extended π system of the synthetic probe.

Dietary exposure to aflatoxins is a significant risk factor in the development of hepatocellular carcinomas. Following bioactivation by microsomal P450s, the reaction of aflatoxin B 1 (AFB 1 ) with guanine (Gua) in DNA leads to the formation of stable, imidazole ring-opened 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B 1 (AFB 1 -FapyGua) adducts. In contrast to most base modifications that result in destabilization of the DNA duplex, the AFB 1 -FapyGua adduct increases the thermal stability of DNA via 5'-interface intercalation and base-stacking interactions. Although it was anticipated that this stabilization might make these lesions difficult to repair relative to helix distorting modifications, prior studies have shown that both the nucleotide and base excision repair pathways participate in the removal of the AFB 1 -FapyGua adduct. Specifically for base excision repair, we previously showed that the DNA glycosylase NEIL1 excises AFB 1 -FapyGua and catalyzes strand scission in both synthetic oligodeoxynucleotides and liver DNA of exposed mice. Since it is anticipated that error-prone replication bypass of unrepaired AFB 1 -FapyGua adducts contributes to cellular transformation and carcinogenesis, the structural and thermodynamic parameters that modulate the efficiencies of these repair pathways are of considerable interest. We hypothesized that the DNA sequence context in which the AFB 1 -FapyGua adduct is formed might modulate duplex stability and consequently alter the efficiencies of NEIL1-initiated repair. To address this hypothesis, site-specific AFB 1 -FapyGua adducts were synthesized in three sequence contexts, with the 5' neighbor nucleotide being varied. DNA structural stability analyses were conducted using UV absorbance- and NMR-based melting experiments. These data revealed differentials in thermal stabilities associated with the 5'-neighbor base pair. Single turnover kinetic analyses using the NEIL1 glycosylase demonstrated corresponding sequence-dependent differences in the repair of this adduct, such that there was an inverse correlation between the stabilization of the duplex and the efficiency of NEIL1-mediated catalysis.

Abasic (AP) sites are one of the most common forms of DNA damage. The deoxyribose ring of AP sites undergoes anomerization between α and β configurations, via an electrophilic aldehyde intermediate. In sequences where an adenine residue is located on the opposing strand and offset 1 nt to the 3' side of the AP site, the nucleophilic N 6 -dA amino group can react with the AP aldehyde residue to form an interstrand cross-link (ICL). Here, we present an experimentally determined structure of the dA-AP ICL by NMR spectroscopy. The ICL was constructed in the oligodeoxynucleotide 5'-d(T 1 A 2 T 3 G 4 T 5 C 6 T 7 A 8 A 9 G 10 T 11 T 12 C 13 A 14 T 15 C 16 T 17 A 18 )-3':5'-d(T 19 A 20 G 21 A 22 T 23 G 24 A 25 A 26 C 27 X 28 T 29 A 30 G 31 A 32 C 33 A 34 T 35 A 36 )-3' (X=AP site), with the dA-AP ICL forming between A 8 and X 28 . The NMR spectra indicated an ordered structure for the cross-linked DNA duplex and afforded detailed spectroscopic resonance assignments. Structural refinement, using molecular dynamics calculations restrained by NOE data (rMD), revealed the structure of the ICL. In the dA-AP ICL, the 2'-deoxyribosyl ring of the AP site was ring-closed and in the β configuration. Juxtapositioning the N 6 -dA amino group and the aldehydic C1 of the AP site within bonding distance while simultaneously maintaining two flanking unpaired A 9 and T 29 bases stacked within the DNA is accomplished by the unwinding of the DNA at the ICL. The structural data is discussed in the context of recent studies describing the replication-dependent unhooking of the dA-AP ICL by the base excision repair glycosylase NEIL3.