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It involves Pol\u03b4-mediated DNA strand displacement synthesis that produces 5\u2032 flaps, FEN1-mediated 5\u2032 flap cleavage, and LIG1-catalyzed nick ligation. Recently, we discovered that under hyperthermal stress conditions, yeast cells convert 5\u2032 flaps into 3\u2032 flaps, which are degraded by 3\u2032 flap nucleases to produce DNA nicks. However, little is known about this 3\u2032 flap-based OFM in human cells. Here, we report that 3\u2032 flaps frequently form in various human cancer cells, and that FEN1 deficiency significantly enhances 3\u2032 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\u2032 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\u2032 flap-based OFM is an important alternative of 5\u2032 flap-based OFM in mammalian cells. XPF is a key nuclease to degrade 3\u2032 flaps and complete OFM for survival. Targeting this compensatory mechanism could provide new therapeutic strategies to selectively impair cancer cell survival under replication stress.<\/jats:p>","DOI":"10.1093\/nar\/gkag536","type":"journal-article","created":{"date-parts":[[2026,5,26]],"date-time":"2026-05-26T07:50:45Z","timestamp":1779781845000},"source":"Crossref","is-referenced-by-count":0,"title":["XPF mediates 3\u2032 flap processing for FEN1-independent Okazaki fragment maturation"],"prefix":"10.1093","volume":"54","author":[{"given":"Kejiao","family":"Li","sequence":"first","affiliation":[{"name":"Department of Cancer Genetics and Epigenetics, City of Hope Beckman Research Institute , Duarte, CA 91010 ,","place":["United States"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Yang","sequence":"additional","affiliation":[{"name":"InnoHK Center for Neuromusculoskeletal Restorative Medicine Limited, Hong Kong Science Park , Hong Kong SAR 999077 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