Upon exposure to endogenous and exogenous alkylating agents, the N2 position of guanine in DNA can be alkylated to yield DNA lesions. Recent studies showed that accurate bypass of various N2-alkyl-dG lesions in mammalian cells require DNA polymerase (Pol) k, where genetic depletion of Pol κ gave rise to significant diminutions in bypass efficiencies for N2-EtdG and N2-nBudG, and induced G → A and G → T mutations. However, it is unclear how Pol k’s function in these processes is modulated by its interactions with other cellular proteins. Herein, we profiled the proximity proteome of Pol k in human cells based on affinity pull-down using CRISPR-engineered HEK293T cells expressing endogenous C-terminally Flag-tagged Pol k in combination with proteomic analysis. We identified several helicases as novel putative Pol k-interacting proteins, suggesting that the polymerase may recruit helicases to unwind unusual DNA structures prior to replicative bypass. We also observed that N2-alkyl-dG lesions could induce R-loop structures, and Pol k can interact with DEAD-box RNA helicase (DDX) 23 to resolve these structures and promote the replicative bypass of these minor-groove DNA lesions. Moreover, our ChIP-seq results revealed that Pol k binds preferentially to chromatin regions that are enriched with R-loop structures. Together, we uncovered that Pol k can interact with DDX23 to facilitate replicative bypass of N2-alkyl-dG lesions, and this process involves DDX23’s role in resolving the lesion-induced R-loop structures.

Pol k interact with DDX23 to unwind R-loop structure induced by N2-alkyl-dG lesions

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ACS Fall 2022

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