Structure of mycobacterial 3′-to-5′ RNA:DNA helicase Lhr bound to a ssDNA tracking strand highlights distinctive features of a novel family of bacterial helicases

Abstract

Mycobacterial Lhr is a DNA damage-inducible superfamily 2 helicase that uses adenosine triphosphate (ATP) hydrolysis to drive unidirectional 3′-to-5′ translocation along single-stranded DNA (ssDNA) and to unwind RNA:DNA duplexes en route. ATPase, translocase and helicase activities are encompassed within the N-terminal 856-amino acid segment. The crystal structure of Lhr-(1–856) in complex with AMPPNP•Mg2+ and ssDNA defines a new helicase family. The enzyme comprises two N-terminal RecA-like modules, a winged helix (WH) domain and a unique C-terminal domain. The 3′ ssDNA end binds in a crescent-shaped groove at the interface between the first RecA domain and the WH domain and tracks 5′ into a groove between the second RecA and C domains. A kissing interaction between the second RecA and C domains forms an aperture that demarcates a putative junction between the loading strand tail and the duplex, with the first duplex nucleoside bookended by stacking on Trp597. Intercalation of Ile528 between nucleosides of the loading strand creates another bookend. Coupling of ATP hydrolysis to RNA:DNA unwinding is dependent on Trp597 and Ile528, and on Thr145 and Arg279 that contact phosphates of the loading strand. The structural and functional data suggest a ratchet mechanism of translocation and unwinding coupled to ATP-driven domain movements.

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

“…Interest in Lhr helicase is driven by its distinctive domain organization and mode of ssDNA binding and the strong induction of its expression after DNA damage in mycobacteria (1)(2)(3)(4). Having solved the crystal structure of the M. smegmatis Lhr-(1-856) (2) and noting the widespread distribution of Lhr-Core homologs in diverse bacteria as part of a gene cluster of putative nucleic acid repair enzymes (Fig.…”

“…2) and seven side chains that contact the ssDNA loading strand (shaded green in Fig. 2) (2). In particular, the four amino acids found to be essential in M. smegmatis Lhr for the coupling of ATP hydrolysis to duplex unwinding (Thr-145, Arg-279, Ile-528, and Trp-597; denoted by triangles in Fig.…”

Mechanistic insights into Lhr helicase function in DNA repair

“…Protein fold, secondary structure and structural homology searches were performed with Phyre2 [ 27 ] under Intensive mode. Predicted structure models were analyzed, superimposed and RMSD calculated with DALI [ 28 ], superimposing against the M. smegmatis Lhr [ 9 ] (PDB: 5V9X) helicase structure. Protein secondary structure was predicted in PSIPRED [ 29 ].…”

“…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 ].…”

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 ., ).…”