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bioRxiv · 10.64898/2026.09.22.753423

Interplay between flap endonuclease 1 and DNA polymerase β in long patch base excision repair: new facets in mechanism

Krasikova, Y. S.·Maltseva, E. A.·Khodyreva, S. N.·Rechkunova, N. I.·Lavrik, O. I.
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Abstract

Mammalian base excision repair (BER) is the major repair pathway for correcting small DNA base lesions. BER operates via two sub-pathways: short patch (or single nucleotide) and long patch BER (SP-BER and LP-BER, respectively). In LP-BER, DNA polymerase beta (Pol{beta}) performs strand-displacement synthesis generating a 5'-flap that is removed by flap endonuclease 1 (FEN1). To determine the precise DNA-intermediate that "switches on" FEN1 activity, we analyzed the FEN1 cleavage position in DNA-intermediates generated by Pol{beta}. For simultaneous detection of Pol{beta}-catalyzed primer elongation and FEN1's cleavage products, we utilized DNA structures with fluorescent labels on the upstream and downstream primers. Our data indicate that FEN1 operates efficiently on DNAs containing one or more equilibrium nucleotides. FEN1 binds to the 3'-terminal equilibrium nucleotide of the primer and probes it for a 3'-OH group. The cleavage point is located directly opposite this 3'-terminal equilibrium nucleotide of the primer. The subsequent elimination of the equilibrium area leads to restoration of the ligatable structure, thereby limiting further strand-displacement synthesis. Since the product sets generated by the two enzymes are mirror-symmetrical, we propose that pausing of Pol{beta} permits FEN1 to join the Pol{beta}-DNA complex. Our study demonstrates that the FEN1-Pol{beta} interplay occurs via the generation and elimination of equilibrium nucleotide areas rather than through a "gap-translation" mechanism. The length of the equilibrium area depends on dNTP concentration, suggesting that the size of the LP-BER repair patches may differ between S-phase and the remainder of the cell cycle.

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Krasikova, Y. S., Maltseva, E. A., Khodyreva, S. N., Rechkunova, N. I., Lavrik, O. I.. 2026-09-23. Interplay between flap endonuclease 1 and DNA polymerase β in long patch base excision repair: new facets in mechanism. https://doi.org/10.64898/2026.09.22.753423

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