Functional Characterization Of Novel RecJ Nucleases With Endonuclease Activity

In this research,RecJ nuclease from Bacillus halodurans or Bacillus alcalophilus(BhRecJ or BaRecJ)had been studied.We performed bioinformatic analyses of the structure and phylogenetic relationships of RecJ nucleases from the above-mentioned strains.Besides,the recombinant expression plasmids were constructed and heterologously expressed in Escherichia coli to study the effects of these class of proteins on host cells in vivo.And these proteins were purified in vitro,to characterize their nuclease activity and to analyze the key residues for the endonuclease activity.So far,all reported RecJs degrade single-stranded DNA(ssDNA)as an exonuclease.In this research,we reported a novel class of RecJ nucleases,represented by BaRecJ and BhRecJ,which possess endo-and exonuclease activities.It is of great significance to further understanding the functional diversity of RecJ proteins.The results are as follow:1.In this research,this novel class of RecJs,represented by BaRecJ and BhRecJ,possess both endo-and exonuclease activities.This class of proteins contain the typical DHH and DHHA1 domains,which are conserved in all RecJs and are responsible for the exonuclease activity,and a novel PIWI-like domain at the C-terminus,which causes the endonuclease activity.As a unique branch,RecJ nucleases with a PIWI-like domain at the C-terminus are widely present in the genus Bacillus.2.Unlike the reported RecJs,this class of RecJs possess endonuclease activity.In vivo,the expression of these proteins caused double-strand breaks(DSBs)in plasmid and genomic DNA in E.coli host cells and resulting in a decrease in the cellular DNA content and cell death.3.In vitro,this novel class of RecJ nucleases can cleave double-stranded DNA(ds DNA)at moderate temperatures.Supercoiled plasmid DNA can be cleaved into open circular and linear forms,and eventually degraded into nucleotides at 23-60°C.The degradation was promoted by Mg²⁺and Mn²⁺.4.Mutational analysis revealed that several important residues affected the endonuclease activity of BaRecJ and BhRecJ.In vitro,the mutation of D561 or E640inhibited the cleavage activity of BaRecJ.And the mutation of D561 inhibited the cleavage activity of BhRecJ.Therefore,the novel C-terminal domain is mainly responsible for endonuclease activity of these proteins.