Mycobacterial helicase Lhr abets resistance to DNA crosslinking agents mitomycin C and cisplatin.

Mycobacterium smegmatis Lhr exemplifies a novel clade of helicases composed of an N-terminal ATPase/helicase domain (Lhr-Core) and a large C-terminal domain (Lhr-CTD) that nucleates a unique homo-tetrameric quaternary structure. Expression of Lhr, and its operonic neighbor Nei2, is induced in mycobacteria exposed to mitomycin C (MMC). Here we report that lhr deletion sensitizes M. smegmatis to killing by DNA crosslinkers MMC and cisplatin but not to killing by monoadduct-forming alkylating agent methyl methanesulfonate or UV irradiation. Testing complementation of MMC and cisplatin sensitivity by expression of Lhr mutants in Δlhr cells established that: (i) Lhr-CTD is essential for DNA repair activity, such that Lhr-Core does not suffice; (ii) ATPase-defective mutant D170A/E171A fails to complement; (iii) ATPase-active, helicase-defective mutant W597A fails to complement and (iv) alanine mutations at the CTD-CTD interface that interdict homo-tetramer formation result in failure to complement. Our results instate Lhr's ATP-driven motor as an agent of inter-strand crosslink repair in vivo, contingent on Lhr's tetrameric quaternary structure. We characterize M. smegmatis Nei2 as a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Counter to previous reports, we find Nei2 is inactive as a lyase at a THF abasic site and has feeble uracil glycosylase activity.

Structure-activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3'-to-5' translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.

Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

Oligomeric quaternary structure of Escherichia coli and Mycobacterium smegmatis Lhr helicases is nucleated by a novel C-terminal domain composed of five winged-helix modules.

Mycobacterium smegmatis Lhr (MsmLhr; 1507-aa) is the founder of a novel clade of bacterial helicases. MsmLhr consists of an N-terminal helicase domain (aa 1-856) with a distinctive tertiary structure (Lhr-Core) and a C-terminal domain (Lhr-CTD) of unknown structure. Here, we report that Escherichia coli Lhr (EcoLhr; 1538-aa) is an ATPase, translocase and ATP-dependent helicase. Like MsmLhr, EcoLhr translocates 3' to 5' on ssDNA and unwinds secondary structures en route, with RNA:DNA hybrid being preferred versus DNA:DNA duplex. The ATPase and translocase activities of EcoLhr inhere to its 877-aa Core domain. Full-length EcoLhr and MsmLhr have homo-oligomeric quaternary structures in solution, whereas their respective Core domains are monomers. The MsmLhr CTD per se is a homo-oligomer in solution. We employed cryo-EM to solve the structure of the CTD of full-length MsmLhr. The CTD protomer is composed of a series of five winged-helix (WH) modules and a ß-barrel module. The CTD adopts a unique homo-tetrameric quaternary structure. A Lhr-CTD subdomain, comprising three tandem WH modules and the ß-barrel, is structurally homologous to AlkZ, a bacterial DNA glycosylase that recognizes and excises inter-strand DNA crosslinks. This homology is noteworthy given that Lhr is induced in mycobacteria exposed to the inter-strand crosslinker mitomycin C.

Structure of a DNA glycosylase that unhooks interstrand cross-links.

DNA glycosylases are important editing enzymes that protect genomic stability by excising chemically modified nucleobases that alter normal DNA metabolism. These enzymes have been known only to initiate base excision repair of small adducts by extrusion from the DNA helix. However, recent reports have described both vertebrate and microbial DNA glycosylases capable of unhooking highly toxic interstrand cross-links (ICLs) and bulky minor groove adducts normally recognized by Fanconi anemia and nucleotide excision repair machinery, although the mechanisms of these activities are unknown. Here we report the crystal structure of Streptomyces sahachiroi AlkZ (previously Orf1), a bacterial DNA glycosylase that protects its host by excising ICLs derived from azinomycin B (AZB), a potent antimicrobial and antitumor genotoxin. AlkZ adopts a unique fold in which three tandem winged helix-turn-helix motifs scaffold a positively charged concave surface perfectly shaped for duplex DNA. Through mutational analysis, we identified two glutamine residues and a ß-hairpin within this putative DNA-binding cleft that are essential for catalytic activity. Additionally, we present a molecular docking model for how this active site can unhook either or both sides of an AZB ICL, providing a basis for understanding the mechanisms of base excision repair of ICLs. Given the prevalence of this protein fold in pathogenic bacteria, this work also lays the foundation for an emerging role of DNA repair in bacteria-host pathogenesis.

Movement of the RecG Motor Domain upon DNA Binding Is Required for Efficient Fork Reversal.

RecG catalyzes reversal of stalled replication forks in response to replication stress in bacteria. The protein contains a fork recognition ("wedge") domain that binds branched DNA and a superfamily II (SF2) ATPase motor that drives translocation on double-stranded (ds)DNA. The mechanism by which the wedge and motor domains collaborate to catalyze fork reversal in RecG and analogous eukaryotic fork remodelers is unknown. Here, we used electron paramagnetic resonance (EPR) spectroscopy to probe conformational changes between the wedge and ATPase domains in response to fork DNA binding by Thermotoga maritima RecG. Upon binding DNA, the ATPase-C lobe moves away from both the wedge and ATPase-N domains. This conformational change is consistent with a model of RecG fully engaged with a DNA fork substrate constructed from a crystal structure of RecG bound to a DNA junction together with recent cryo-electron microscopy (EM) structures of chromatin remodelers in complex with dsDNA. We show by mutational analysis that a conserved loop within the translocation in RecG (TRG) motif that was unstructured in the RecG crystal structure is essential for fork reversal and DNA-dependent conformational changes. Together, this work helps provide a more coherent model of fork binding and remodeling by RecG and related eukaryotic enzymes.

Structure and in vivo psoralen DNA crosslink repair activity of mycobacterial Nei2.

Mycobacterium smegmatis Nei2 is a monomeric enzyme with AP ß-lyase activity on single-stranded DNA. Expression of Nei2, and its operonic neighbor Lhr (a tetrameric 3'-to-5' helicase), is induced in mycobacteria exposed to DNA damaging agents. Here, we find that nei2 deletion sensitizes M. smegmatis to killing by DNA inter-strand crosslinker trimethylpsoralen but not to crosslinkers mitomycin C and cisplatin. By contrast, deletion of lhr sensitizes to killing by all three crosslinking agents. We report a 1.45 Å crystal structure of recombinant Nei2, which is composed of N and C terminal lobes flanking a central groove suitable for DNA binding. The C lobe includes a tetracysteine zinc complex. Mutational analysis identifies the N-terminal proline residue (Pro2 of the ORF) and Lys51, but not Glu3, as essential for AP lyase activity. We find that Nei2 has 5-hydroxyuracil glycosylase activity on single-stranded DNA that is effaced by alanine mutations of Glu3 and Lys51 but not Pro2. Testing complementation of psoralen sensitivity by expression of wild-type and mutant nei2 alleles in ∆nei2 cells established that AP lyase activity is neither sufficient nor essential for crosslink repair. By contrast, complementation of psoralen sensitivity of ∆lhr cells by mutant lhr alleles depended on Lhr's ATPase/helicase activities and its tetrameric quaternary structure. The lhr-nei2 operon comprises a unique bacterial system to rectify inter-strand crosslinks.IMPORTANCEThe DNA inter-strand crosslinking agents mitomycin C, cisplatin, and psoralen-UVA are used clinically for the treatment of cancers and skin diseases; they have been invaluable in elucidating the pathways of inter-strand crosslink repair in eukaryal systems. Whereas DNA crosslinkers are known to trigger a DNA damage response in bacteria, the roster of bacterial crosslink repair factors is incomplete and likely to vary among taxa. This study implicates the DNA damage-inducible mycobacterial lhr-nei2 gene operon in protecting Mycobacterium smegmatis from killing by inter-strand crosslinkers. Whereas interdicting the activity of the Lhr helicase sensitizes mycobacteria to mitomycin C, cisplatin, and psoralen-UVA, the Nei2 glycosylase functions uniquely in evasion of damage caused by psoralen-UVA.