Characterization of Lhr-Core DNA helicase and manganese- dependent DNA nuclease components of a bacterial gene cluster encoding nucleic acid repair enzymes.

Lhr is a large superfamily 2 helicase present in mycobacteria and a moderate range of other bacterial taxa. A shorter version of Lhr, here referred to as Lhr-Core, is distributed widely in bacteria, where it is often encoded in a gene cluster along with predicted binuclear metallo-phosphoesterase (MPE), ATP-dependent DNA ligase, and metallo-β-lactamase exonuclease enzymes. Here we characterized the Lhr-Core and MPE proteins from Pseudomonas putida We report that P. putida Lhr-Core is an ssDNA-dependent ATPase/dATPase (Km , 0.37 mm ATP; k cat, 3.3 s-1), an ATP-dependent 3'-to-5' single-stranded DNA translocase, and an ATP-dependent 3'-to-5' helicase. Lhr-Core unwinds 3'-tailed duplexes in which the loading/tracking strand is DNA and the displaced strand is either DNA or RNA. We found that P. putida MPE is a manganese-dependent phosphodiesterase that releases p-nitrophenol from bis-p-nitrophenyl phosphate (k cat, 212 s-1) and p-nitrophenyl-5'-thymidylate (k cat, 34 s-1) but displays no detectable phosphomonoesterase activity against p-nitrophenyl phosphate. MPE is also a manganese-dependent DNA endonuclease that sequentially converts a closed-circle plasmid DNA to nicked circle and linear forms prior to degrading the linear DNA to produce progressively smaller fragments. The biochemical activities of MPE and a structure predicted in Phyre2 point to MPE as a new bacterial homolog of Mre11. Genetic linkage of a helicase and DNA nuclease with a ligase and a putative exonuclease (a predicted homolog of the SNM1/Apollo family of nucleases) suggests that these enzymes comprise or participate in a bacterial DNA repair pathway.