Abstract
Background
All RecJ proteins are known to date only perform exonuclease activity. The present study reports that a novel RecJ protein obtained from Bacillus cereus isolated from marine sediments has both endonuclease and exonuclease activities.
Methods
Analysis of the BcRecJ expression induction in E. coli BL21 revealed that the BcRecJ protein cleaved plasmids and genomic DNA in the host cell, and led to cell death and decreased the DNA content. Further, the BcRecJ protein had the ability to degrade supercoiled plasmid DNA into circular or linear forms in vitro. Meanwhile, the BcRecJ protein loaded with an S-modified guide facilitated plasmid linearization and reduced smear formation.
Results
The results suggested that this novel BcRecJ protein was different from any reported RecJs and had a longer C-terminus. Testing the BcRecJ mutants indicated that the endonuclease activity was affected by two residues of BcRecJ (D561, E637) after testing the BcRecJ mutants.
Conclusion
The discovery of the type of protein is a new breakthrough for the RecJ proteins, which has both endonuclease and exonuclease activities.
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Acknowledgements
This work was supported by National Key R&D Program of China (Grant 2018YFC0310701) and Deep Sea Biological Resources Program (No. DY135-B2-11).
Supplementary Information
Supplemental Table 1—The ratio of plasmid and genomic DNA content. Supplemental Table 2—Primers and plasmids used in this experiment. Supplemental Table 3—Oligonucleotides used to characterize the nuclease activity. Supplemental Table 4—Oligos used for nuclease activity of BcRecJ. Supplemental Figure 1a—Sequence alignment of RecJ in Bacillus cereus with sequences of RecJs in Escherichia coli and Thermus thermophilus. Conserved domains are highlighted with frames. Supplemental Figure 1b—Phylogenetic tree of bacterial RecJs. The bootstrap values are listed at the branch points. Supplemental Figure 2—Purity analysis of BcRecJ and mutants by 15% SDS-PAGE. Supplemental Figure 3a —The activity of BcRecJ (500 nM BaRecJ, 750 ng plasmid DNA, cleavage for 2 h at 37 °C) in the presence of 1 mM Mg2+, Mn2+, Ca2+, Cu2+, Li2+, Ni2+, or Co2+. Lane 2, pUC19-s plasmid control. Supplemental Figure 3b—Effect of different Mg2+ concentrations in buffer solution on the activity of BcRecJ toward pUC19-s at 37 °C for 2 h. Lane 2, pUC19-s plasmid control. Supplemental Figure 3c—Effect of different Na+ concentrations in buffer solution on the activity of BcRecJ toward pUC19-s at 37 °C for 2 h. Lane 2, pUC19-s plasmid control.
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Liya Ma declares that she has no conflict of interest. Wen Wang declares that she has no conflict of interest. Chaozhi Hao sdeclares that he has no conflict of interest. Li Zheng declares that he has no conflict of interest. Ling Wang declares that she has no conflict of interest. Minggang Zheng declares that he has no conflict of interest.
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Ma, L., Wang, W., Hao, C. et al. Coexistence of endonuclease and exonuclease activities in a novel RecJ from Bacillus cereus. Biotechnol Lett 43, 1349–1355 (2021). https://doi.org/10.1007/s10529-021-03107-z
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DOI: https://doi.org/10.1007/s10529-021-03107-z