N2-alkyl-dG lesions trigger R-loop formation in genome
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PROVIDER: PRJNA1042825 | ENA |
REPOSITORIES: ENA
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Project description:Strand-specific kethoxal-assisted single-stranded DNA (ssDNA) sequencing (spKAS-seq) for mapping R-loop formation in HEK293T cells with/without the N2-heptynyl-dG treatment .
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Project description:DNA alkylation at the N2 position of guanine (N2-dG) is a prevalent type of DNA minor-groove lesions arising from various exogenous environmental contaminants and endogenous cellular processes. These N2-alkyl-dG lesions can induce G → T mutations during transcription if left unrepaired. However, the repair pathways of N2-alkyl-dG lesions remain incompletely elucidated. In this study, we identified a series of potential N2-alkyl-dG-binding proteins utilizing an affinity pulldown coupled with quantitative proteomic approach. We investigated their roles in DNA damage response and repair of these lesions. High-mobility group protein B3 (HMGB3) and activated RNA polymerase II transcriptional coactivator P15 (SUB1) exhibited preferential binding toward N2-nBudG-containing duplex DNA in quantitative proteomics analysis and in vitro binding assay using recombinant proteins. The presence of HMGB3 and SUB1 protected cells against alkylating agents (e.g., BPDE). Both HMGB3 and SUB1 modulated the repair of N2-nBudG and trans-N2-BPDE-dG in genomic DNA, while HMGB3 wasn’t involved in the repair of cis-N2-BPDE-dG. Together, our findings provided new knowledge about the cellular sensing and repair of minor-groove N2-alkyl-dG lesions.
2024-08-02 | PXD051678 | Pride
Project description:N2-heptynyl-dG sequencing in HEK293T cells
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Project description:Michael addition of trans-4-hydroxynonenal (HNE) to deoxyguanosine yields diastereomeric 1,N(2)-dG adducts in DNA. When placed opposite dC in the 5'-CpG-3' sequence, the (6S,8R,11S) diastereomer forms a N(2)-dG:N(2)-dG interstrand cross-link [Wang, H.; Kozekov, I. D.; Harris, T. M.; Rizzo, C. J. J. Am. Chem. Soc.2003, 125, 5687-5700]. We refined its structure in 5'-d(G(1)C(2)T(3)A(4)G(5)C(6)X(7)A(8)G(9)T(10)C(11)C(12))-3'·5'-d(G(13)G(14)A(15)C(16)T(17)C(18)Y(19)C(20)T(21)A(22)G(23)C(24))-3' [X(7) is the dG adjacent to the C6 carbon of the cross-link or the α-carbon of the (6S,8R,11S) 1,N(2)-dG adduct, and Y(19) is the dG adjacent to the C8 carbon of the cross-link or the γ-carbon of the HNE-derived (6S,8R,11S) 1,N(2)-dG adduct; the cross-link is in the 5'-CpG-3' sequence]. Introduction of (13)C at the C8 carbon of the cross-link revealed one (13)C8→H8 correlation, indicating that the cross-link existed predominantly as a carbinolamine linkage. The H8 proton exhibited NOEs to Y(19) H1', C(20) H1', and C(20) H4', orienting it toward the complementary strand, consistent with the (6S,8R,11S) configuration. An NOE was also observed between the HNE H11 proton and Y(19) H1', orienting the former toward the complementary strand. Imine and pyrimidopurinone linkages were excluded by observation of the Y(19)N(2)H and X(7) N1H protons, respectively. A strong H8→H11 NOE and no (3)J((13)C→H) coupling for the (13)C8-O-C11-H11 eliminated the tetrahydrofuran species derived from the (6S,8R,11S) 1,N(2)-dG adduct. The (6S,8R,11S) carbinolamine linkage and the HNE side chain were located in the minor groove. The X(7)N(2) and Y(19)N(2) atoms were in the gauche conformation with respect to the linkage, maintaining Watson-Crick hydrogen bonds at the cross-linked base pairs. A solvated molecular dynamics simulation indicated that the anti conformation of the hydroxyl group with respect to C6 of the tether minimized steric interaction and predicted hydrogen bonds involving O8H with C(20)O(2) of the 5'-neighbor base pair G(5)·C(20) and O11H with C(18)O(2) of X(7)·C(18). These may, in part, explain the stability of this cross-link and the stereochemical preference for the (6S,8R,11S) configuration.
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Project description:Endogenous metabolites and exogenous chemicals can induce covalent modifications on DNA, producing DNA lesions. The N2 of guanine was shown to be a common alkylation site in DNA; however, not much is known about the influence of the size of the alkyl group in N2-alkyldG lesions on cellular DNA replication or how translesion synthesis (TLS) polymerases modulate DNA replication past these lesions in human cells. To answer these questions, we employ a robust shuttle vector method to investigate the impact of four N2-alkyldG lesions (i.e., with the alkyl group being a methyl, ethyl, n-propyl, or n-butyl group) on DNA replication in human cells. We find that replication through the N2-alkyldG lesions was highly efficient and accurate in HEK293T cells or isogenic CRISPR-engineered cells with deficiency in polymerase (Pol) ? or Pol ?. Genetic ablation of Pol ?, Pol ?, or Rev1, however, results in decreased bypass efficiencies and elicits substantial frequencies of G ? A transition and G ? T transversion mutations for these lesions. Moreover, further depletion of Pol ? in Pol ?- or Pol ?-deficient cells gives rise to elevated rates of G ? A and G ? T mutations and substantially decreased bypass efficiencies. Cumulatively, we demonstrate that the error-free replication past the N2-alkyldG lesions is facilitated by a specific subset of TLS polymerases, and we find that longer alkyl chains in these lesions induce diminished bypass efficiency and fidelity in DNA replication.
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