Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids

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The current model for Okazaki fragment maturation in bacteria invokes RNA cleavage by RNase H, followed by strand displacement synthesis and 50 RNA flap removal by DNA polymerase I (Pol I). RNA removal by Pol I is thought to occur through the 50-30 flap endo/exonuclease (FEN) domain, located in the N-terminus of the protein. In addition to Pol I, many bacteria encode a second, Pol I-independent FEN. The contribution of Pol I and Pol I-independent FENs to DNA replication and genome stability remains unclear. In this work we purified Bacillus subtilis Pol I and FEN, then assayed these proteins on a variety of RNA-DNA hybrid and DNA-only substrates. We found that FEN is far more active than Pol I on nicked double-flap, 50 single flap, and nicked RNA-DNA hybrid substrates. We show that the 50 nuclease activity of B. subtilis Pol I is feeble, even during DNA synthesis when a 50 flapped substrate is formed modeling an Okazaki fragment intermediate. Examination of Pol I and FEN on DNA-only substrates shows that FEN is more active than Pol I on most substrates tested. Further experiments show that ΔpolA phenotypes are completely rescued by expressing the C-terminal polymerase domain while expression of the N-terminal 50 nuclease domain fails to complement ΔpolA. Cells lacking FEN (ΔfenA) show a phenotype in conjunction with an RNase HIII defect, providing genetic evidence for the involvement of FEN in Okazaki fragment processing. With these results, we propose a model where cells remove RNA primers using FEN while upstream Okazaki fragments are extended through synthesis by Pol I. Our model resembles Okazaki fragment processing in eukaryotes, where Pol δ catalyzes strand displacement synthesis followed by 50 flap cleavage using FEN-1. Together our work highlights the conservation of ordered steps for Okazaki fragment processing in cells ranging from bacteria to human.




Lowder, F. C., & Simmons, L. A. (2023). Bacillus subtilis encodes a discrete flap endonuclease that cleaves RNA-DNA hybrids. PLoS Genetics, 19(5).

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