XPF mediates 3' flap processing for FEN1-independent Okazaki fragment maturation.

Okazaki fragment maturation (OFM), the process that removes RNA-DNA primers, is a major source of DNA replication stress and mutations. It involves Polδ-mediated DNA strand displacement synthesis that produces 5' flaps, FEN1-mediated 5' flap cleavage, and LIG1-catalyzed nick ligation. Recently, we discovered that under hyperthermal stress conditions, yeast cells convert 5' flaps into 3' flaps, which are degraded by 3' flap nucleases to produce DNA nicks. However, little is known about this 3' flap-based OFM in human cells. Here, we report that 3' flaps frequently form in various human cancer cells, and that FEN1 deficiency significantly enhances 3' flap levels. XPF1 is recruited to the replication forks in FEN1 mutant or FEN1-chemically inhibited cells. Notably, XPF deficiency or inhibition in those defective cells leads to accumulation of 3' flaps, replication-related DNA strand breaks, and unique mutation signatures. Furthermore, XPF and FEN1 inhibitors show synergistic effects in killing human cancer cells. In summary, we demonstrate that 3' flap-based OFM is an important alternative of 5' flap-based OFM in mammalian cells. XPF is a key nuclease to degrade 3' flaps and complete OFM for survival. Targeting this compensatory mechanism could provide new therapeutic strategies to selectively impair cancer cell survival under replication stress.