APOBEC3C coordinates DDX5 in R-loop resolution and dynamic control of Chk1-mediated stress-responsive circuitry as a prerequisite for gemcitabine resistance in p53-deficient cells.

Genomic instability is a hallmark of cancer, encompassing both sequence and structural alterations that drive tumor evolution and heterogeneity. The APOBEC3 family of deoxycytidine deaminases has emerged as a major source of mutagenic activity in cancers. R-loops are RNA-DNA hybrids and structural barriers that interfere with replication and transcription. Among the APOBEC3 family, APOBEC3C (A3C) is particularly worthy of attention for its upregulation, driving the DNA replication stress tolerance in response to replication stress-inducing drug gemcitabine. However, the molecular mechanisms of gemcitabine resistance and regulatory circuitries mediated by A3C remain largely unknown, especially in checkpoint-deficient tumors. Initially, we screened that A3C was a putative transcriptional target of p53, and p53-deficient H1299 cells harboring A3C elicited a chemoresistant phenotype upon gemcitabine treatment both in vitro and in vivo. A3C expression enhanced Chk1-dependent S-phase checkpoint activation, thus slowing down replication fork progression and facilitating DNA repair. Pull-down assay and proteomic analysis identified that A3C had a specific interaction with the RNA helicase DDX5, which coordinately played critical roles in R-loop resolution. In contrast to A3C, DDX5 expression attenuated Chk1-dependent S-phase checkpoint activation. Knockdown of DDX5 in A3C-proficient H1299 cells attenuated gemcitabine-induced Chk1 activation and enhanced the therapeutic index of gemcitabine by promoting R-loop accumulation. Therefore, we conclude that A3C/DDX5/R-loop complex may impair the sensitivity of gemcitabine by modulating Chk1 dynamics and DNA replication/damage response machinery.