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

Abstract

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.