Functional characterization of Mrr-family nuclease SLL1429 involved in MMC and phage resistance

Abstract

Cyanobacteria, autotrophic prokaryotes capable of oxygenic photosynthesis, are important atmospheric carbon fixers of Earth and potential alternatives for producing green fuels and chemicals. However, they face significant environmental stress during growth, such as Ultraviolet radiation, salt, and cyanophage exposure, which can impact their physiology and growth. Nucleases, such as Mrr (Methylated adenine Recognition and Restriction) endonuclease, play key roles in stress response, DNA repair, or anti-phage functions, but these in cyanobacteria remains underexplored. The SLL1429 protein with Mrr/NA-iREase1 domain was predicted to play a role as a nuclease in stress resistance in cyanobacteria. In this study, our findings indicate that SLL1429 is a PD-(D/E)XK superfamily nuclease with DNase activities towards various DNA structures, including dsDNA, Holliday junction, Flap and Flap derivatives. The nuclease activity of SLL1429 is dependent on the Mrr domain. However, unlike classic Mrr, SLL1429 recognizes and cleaves both methylated and unmethylated DNA substrates. Notably, SLL1429 plays a role in Mitomycin C (MMC) resistance in Synechocystis sp. PCC6803 and anti-phage activity in E. coli. In view of the above, SLL1429 of Synechocystis sp. PCC6803 has been identified as a new stress-resistant nuclease. This discovery provides novel perspectives on the mechanism of environmental adaption in cyanobacteria and lays a theoretical foundation for further exploration of "microbial cell factory".