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

Abstract

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 We report that Lhr-Core is an ssDNA-dependent ATPase/dATPase ( , 0.37 mm ATP; , 3.3 s), 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 MPE is a manganese-dependent phosphodiesterase that releases-nitrophenol from bis--nitrophenyl phosphate ( , 212 s) and -nitrophenyl-5'-thymidylate ( , 34 s) but displays no detectable phosphomonoesterase activity against -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.

Mechanistic insights into Lhr helicase function in DNA repair

“…The bacterial ‘core’ Lhr (Lhr-Core), which lacks a 700 amino acid C-terminal region present in the bacterial but not archaeal Lhr enzymes, is a ssDNA-stimulated ATPase that translocates ssDNA with 3′ to 5′ directionality [ 10 ]. Purified full-length archaeal Lhr ( Supplementary Figure S1 ) was challenged with a gapped DNA duplex substrate to determine if it had similar properties ( Figure 2A ).…”

“…Bacterial Lhr is extended to 1300–1500 amino acids by a region of unknown function that lacks obvious sequence homologues. Biochemical analysis of the Lhr-Core from the bacteria Mycobacterium smegmatis and Pseudomonas putida identified ATP-dependent ssDNA translocation with 3′ to 5′ directionality [ 1 , 9 , 10 ]. A crystal structure of bacterial Lhr-Core highlights significant similarities with the archaeal DNA repair helicase Hel308 [ 9 , 11 ], most notably in the orientation and interaction of its winged helix domain (WHD) with RecA-like domains typical of Ski2-like helicases [ 12 , 13 ].…”

Structural insight into DNA joining: from conserved mechanisms to diverse scaffolds

“…Lig B ligases appear in gene clusters with a novel Lhr-helicase, binuclear metallophosphoesterase (MPE) and putative exonuclease. Lhr is an ATP-dependent 3′ to 5′ helicase which unwinds DNA–DNA and DNA–RNA duplexes preceded by a single-stranded loading segment ( 54 ). MPE is a Mn dependent single-strand endonuclease that cleaves both linear and loops of stem-and-loop structures ( 55 ).…”

“…MPE is a Mn dependent single-strand endonuclease that cleaves both linear and loops of stem-and-loop structures ( 55 ). The exonuclease member of the cluster has not yet been biochemically characterized, but it is predicted to be a homolog of the SNM1B/Apollo nuclease which repairs inter-strand crosslinks ( 54 ). Although the precise biological substrate and order of activity have not yet been determined, it is likely that these enzymes represent yet another distinct repair pathway in bacteria (Figure 8B ).…”

Omnipresent Maxwell's demons orchestrate information management in living cells

“…In the same vein (Table ), several other E. coli ATP‐dependent helicases that may act as MxDs for discrimination of specific targets are also involved in a variety of recombination processes. Lhr(RhlF) is a large helicase of unknown specificity (Ejaz and Shuman, ) also active in M. tuberculosis , possibly involved in site‐specific recombination (Ejaz et al ., ). HelC(YoaA) directs repair factors to a blocked DNA fork (Brown et al ., ).…”