Structure and mechanism of Mycobacterium smegmatis polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of M. tuberculosis militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of Mycobacterium smegmatis PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.

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.

Caveat mutator: alanine substitutions for conserved amino acids in RNA ligase elicit unexpected rearrangements of the active site for lysine adenylylation.

Naegleria gruberi RNA ligase (NgrRnl) exemplifies the Rnl5 family of adenosine triphosphate (ATP)-dependent polynucleotide ligases that seal 3'-OH RNA strands in the context of 3'-OH/5'-PO4 nicked duplexes. Like all classic ligases, NgrRnl forms a covalent lysyl-AMP intermediate. A two-metal mechanism of lysine adenylylation was established via a crystal structure of the NgrRnl•ATP•(Mn2+)2 Michaelis complex. Here we conducted an alanine scan of active site constituents that engage the ATP phosphates and the metal cofactors. We then determined crystal structures of ligase-defective NgrRnl-Ala mutants in complexes with ATP/Mn2+. The unexpected findings were that mutations K170A, E227A, K326A and R149A (none of which impacted overall enzyme structure) triggered adverse secondary changes in the active site entailing dislocations of the ATP phosphates, altered contacts to ATP, and variations in the numbers and positions of the metal ions that perverted the active sites into off-pathway states incompatible with lysine adenylylation. Each alanine mutation elicited a distinctive off-pathway distortion of the ligase active site. Our results illuminate a surprising plasticity of the ligase active site in its interactions with ATP and metals. More broadly, they underscore a valuable caveat when interpreting mutational data in the course of enzyme structure-function studies.

Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.

Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.

Structures of ATP-bound DNA ligase D in a closed domain conformation reveal a network of amino acid and metal contacts to the ATP phosphates.

DNA ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA ligases join 3'-OH and 5'-PO4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and release pyrophosphate (PPi) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA ligases are ATP-dependent. The catalytic core of ATP-dependent DNA ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4-1.8 Å resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (∼50 Å), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PPi leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg2+ that engages the ATP α phosphate and a second metal that bridges the ATP ß and γ phosphates.

Two-metal versus one-metal mechanisms of lysine adenylylation by ATP-dependent and NAD+-dependent polynucleotide ligases.

Polynucleotide ligases comprise a ubiquitous superfamily of nucleic acid repair enzymes that join 3'-OH and 5'-PO4 DNA or RNA ends. Ligases react with ATP or NAD+ and a divalent cation cofactor to form a covalent enzyme-(lysine-Nζ)-adenylate intermediate. Here, we report crystal structures of the founding members of the ATP-dependent RNA ligase family (T4 RNA ligase 1; Rnl1) and the NAD+-dependent DNA ligase family (Escherichia coli LigA), captured as their respective Michaelis complexes, which illuminate distinctive catalytic mechanisms of the lysine adenylylation reaction. The 2.2-Å Rnl1•ATP•(Mg2+)2 structure highlights a two-metal mechanism, whereby: a ligase-bound "catalytic" Mg2+(H2O)5 coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; a second octahedral Mg2+ coordination complex bridges the ß and γ phosphates; and protein elements unique to Rnl1 engage the γ phosphate and associated metal complex and orient the pyrophosphate leaving group for in-line catalysis. By contrast, the 1.55-Å LigA•NAD+•Mg2+ structure reveals a one-metal mechanism in which a ligase-bound Mg2+(H2O)5 complex lowers the lysine pKa and engages the NAD+ α phosphate, but the ß phosphate and the nicotinamide nucleoside of the nicotinamide mononucleotide (NMN) leaving group are oriented solely via atomic interactions with protein elements that are unique to the LigA clade. The two-metal versus one-metal dichotomy demarcates a branchpoint in ligase evolution and favors LigA as an antibacterial drug target.

Homologous recombination mediated by the mycobacterial AdnAB helicase without end resection by the AdnAB nucleases.

Current models of bacterial homologous recombination (HR) posit that extensive resection of a DNA double-strand break (DSB) by a multisubunit helicase-nuclease machine (e.g. RecBCD, AddAB or AdnAB) generates the requisite 3' single-strand DNA substrate for RecA-mediated strand invasion. AdnAB, the helicase-nuclease implicated in mycobacterial HR, consists of two subunits, AdnA and AdnB, each composed of an N-terminal ATPase domain and a C-terminal nuclease domain. DSB unwinding by AdnAB in vitro is stringently dependent on the ATPase activity of the 'lead' AdnB motor translocating on the 3' ssDNA strand, but not on the putative 'lagging' AdnA ATPase. Here, we queried genetically which activities of AdnAB are pertinent to its role in HR and DNA damage repair in vivo by inactivating each of the four catalytic domains. Complete nuclease-dead AdnAB enzyme can sustain recombination in vivo, as long as its AdnB motor is intact and RecO and RecR are available. We conclude that AdnAB's processive DSB unwinding activity suffices for AdnAB function in HR. Albeit not excluding the agency of a backup nuclease, our findings suggest that mycobacterial HR can proceed via DSB unwinding and protein capture of the displaced 3'-OH single strand, without a need for extensive end-resection.

Crystal Structure and Biochemical Characterization of a Mycobacterium smegmatis AAA-Type Nucleoside Triphosphatase Phosphohydrolase (Msm0858).

UNLABELLED: AAA proteins (ATPases associated with various cellular activities) use the energy of ATP hydrolysis to drive conformational changes in diverse macromolecular targets. Here, we report the biochemical characterization and 2.5-Å crystal structure of a Mycobacterium smegmatis AAA protein Msm0858, the ortholog of Mycobacterium tuberculosis Rv0435c. Msm0858 is a magnesium-dependent ATPase and is active with all nucleoside triphosphates (NTPs) and deoxynucleoside triphosphates (dNTPs) as substrates. The Msm0858 structure comprises (i) an N-terminal domain (amino acids [aa] 17 to 201) composed of two ß-barrel modules and (ii) two AAA domains, D1 (aa 212 to 473) and D2 (aa 476 to 744), each of which has ADP in the active site. Msm0858-ADP is a monomer in solution and in crystallized form. Msm0858 domains are structurally homologous to the corresponding modules of mammalian p97. However, the position of the N-domain modules relative to the AAA domains in the Msm0858-ADP tertiary structure is different and would impede the formation of a p97-like hexameric quaternary structure. Mutational analysis of the A-box and B-box motifs indicated that the D1 and D2 AAA domains are both capable of ATP hydrolysis. Simultaneous mutations of the D1 and D2 active-site motifs were required to abolish ATPase activity. ATPase activity was effaced by mutation of the putative D2 arginine finger, suggesting that Msm0858 might oligomerize during the ATPase reaction cycle. A truncated variant Msm0858 (aa 212 to 745) that lacks the N domain was characterized as a catalytically active homodimer. IMPORTANCE: Recent studies have underscored the importance of AAA proteins (ATPases associated with various cellular activities) in the physiology of mycobacteria. This study reports the ATPase activity and crystal structure of a previously uncharacterized mycobacterial AAA protein, Msm0858. Msm0858 consists of an N-terminal ß-barrel domain and two AAA domains, each with ADP bound in the active site. Msm0858 is a structural homolog of mammalian p97, with respect to the linear order and tertiary structures of their domains.

Structure and two-metal mechanism of a eukaryal nick-sealing RNA ligase.

ATP-dependent RNA ligases are agents of RNA repair that join 3'-OH and 5'-PO4 RNA ends. Naegleria gruberi RNA ligase (NgrRnl) exemplifies a family of RNA nick-sealing enzymes found in bacteria, viruses, and eukarya. Crystal structures of NgrRnl at three discrete steps along the reaction pathway-covalent ligase-(lysyl-Nζ)-AMP•Mn(2+) intermediate; ligase•ATP•(Mn(2+))2 Michaelis complex; and ligase•Mn(2+) complex-highlight a two-metal mechanism of nucleotidyl transfer, whereby (i) an enzyme-bound "catalytic" metal coordination complex lowers the pKa of the lysine nucleophile and stabilizes the transition state of the ATP α phosphate; and (ii) a second metal coordination complex bridges the ß- and γ-phosphates. The NgrRnl N domain is a distinctively embellished oligonucleotide-binding (OB) fold that engages the γ-phosphate and associated metal complex and orients the pyrophosphate leaving group for in-line catalysis with stereochemical inversion at the AMP phosphate. The unique domain architecture of NgrRnl fortifies the theme that RNA ligases have evolved many times, and independently, by fusions of a shared nucleotidyltransferase domain to structurally diverse flanking modules. The mechanistic insights to lysine adenylylation gained from the NgrRnl structures are likely to apply broadly to the covalent nucleotidyltransferase superfamily of RNA ligases, DNA ligases, and RNA capping enzymes.

Characterization of a novel eukaryal nick-sealing RNA ligase from Naegleria gruberi.

The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes, including 15 putative RNA ligases, one of which, NgrRnl, is a eukaryal homolog of Deinococcus radiodurans RNA ligase, DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3'-OH/5'-PO4 duplexes in which the 3'-OH strand is RNA. It does so via the "classic" ligase pathway, entailing reaction with ATP to form a covalent NgrRnl-AMP intermediate, transfer of AMP to the nick 5'-PO4, and attack of the RNA 3'-OH on the adenylylated nick to form a 3'-5' phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases, NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead, it contains a defining amino-terminal domain that we show is important for 3'-OH/5'-PO4 nick-sealing and ligase adenylylation, but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl, DraRnl, and their homologs from diverse bacteria, viruses, and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.

Discrimination of RNA from DNA by polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also thought to participate in bacterial DNA transactions. Here we used chimeric polynucleotides, composed of alternating RNA and DNA tracts, to analyze whether and how Mycobacterium smegmatis PNPase discriminates RNA from DNA during the 3'-phosphorolysis reaction. We find that a kinetic block to 3'-phosphorolysis of a DNA tract within an RNA polynucleotide is exerted when resection has progressed to the point that a 3'-monoribonucleotide flanks the impeding DNA segment. The position of the pause one nucleotide before the first deoxynucleotide encountered is independent of DNA tract length. However, the duration of the pause is affected by DNA tract length, being transient for a single deoxynucleotide and durable when two or more consecutive deoxynucleotides are encountered. Substituting manganese for magnesium as the metal cofactor allows PNPase to "nibble" into the DNA tract. A 3'-phosphate group prevents RNA phosphorolysis when the metal cofactor is magnesium. With manganese, PNPase can resect an RNA 3'-phosphate end, albeit 80-fold slower than a 3'-OH. We discuss the findings in light of the available structures of PNPase and the archaeal exosome·RNA·phosphate complex and propose a model for catalysis whereby the metal cofactor interacts with the scissile phosphodiester and the penultimate ribose.

Distinctive effects of domain deletions on the manganese-dependent DNA polymerase and DNA phosphorylase activities of Mycobacterium smegmatis polynucleotide phosphorylase.

Polynucleotide phosphorylase (PNPase) plays synthetic and degradative roles in bacterial RNA metabolism; it is also suggested to participate in bacterial DNA transactions. Here we characterize and compare the RNA and DNA modifying activities of Mycobacterium smegmatis PNPase. The full-length (763-aa) M. smegmatis PNPase is a homotrimeric enzyme with Mg(2+)•PO(4)-dependent RNA 3'-phosphorylase and Mg(2+)•ADP-dependent RNA polymerase activities. We find that the enzyme is also a Mn(2+)•dADP-dependent DNA polymerase and a Mn(2+)•PO(4)-dependent DNA 3'-phosphorylase. The Mn(2+)•DNA and Mg(2+)•RNA end modifying activities of mycobacterial PNPase are coordinately ablated by mutating the putative manganese ligand Asp526, signifying that both metals likely bind to the same site on PNPase. Deletions of the C-terminal S1 and KH domains of mycobacterial PNPase exert opposite effects on the RNA and DNA modifying activities. Subtracting the S1 domain diminishes RNA phosphorylase and polymerase activity; simultaneous deletion of the S1 and KH domains further cripples the enzyme with respect to RNA substrates. By contrast, the S1 and KH domain deletions enhance the DNA polymerase and phosphorylase activity of mycobacterial PNPase. We observe two distinct modes of nucleic acid binding by mycobacterial PNPase: (i) metal-independent RNA-specific binding via the S1 domain, and (ii) metal-dependent binding to RNA or DNA that is optimal when the S1 domain is deleted. These findings add a new dimension to our understanding of PNPase specificity, whereby the C-terminal modules serve a dual purpose: (i) to help capture an RNA polynucleotide substrate for processive 3' end additions or resections, and (ii) to provide a specificity filter that selects against a DNA polynucleotide substrate.

A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing.

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.

Mycobacterium smegmatis RqlH defines a novel clade of bacterial RecQ-like DNA helicases with ATP-dependent 3'-5' translocase and duplex unwinding activities.

The Escherichia coli RecQ DNA helicase participates in a pathway of DNA repair that operates in parallel to the recombination pathway driven by the multisubunit helicase-nuclease machine RecBCD. The model mycobacterium Mycobacterium smegmatis executes homologous recombination in the absence of its helicase-nuclease machine AdnAB, though it lacks a homolog of E. coli RecQ. Here, we identify and characterize M. smegmatis RqlH, a RecQ-like helicase with a distinctive domain structure. The 691-amino acid RqlH polypeptide consists of a RecQ-like ATPase domain (amino acids 1-346) and tetracysteine zinc-binding domain (amino acids 435-499), separated by an RqlH-specific linker. RqlH lacks the C-terminal HRDC domain found in E. coli RecQ. Rather, the RqlH C-domain resembles bacterial ComF proteins and includes a phosphoribosyltransferase-like module. We show that RqlH is a DNA-dependent ATPase/dATPase that translocates 3'-5' on single-stranded DNA and has 3'-5' helicase activity. These functions inhere to RqlH-(1-505), a monomeric motor unit comprising the ATPase, linker and zinc-binding domains. RqlH homologs are distributed widely among bacterial taxa. The mycobacteria that encode RqlH lack a classical RecQ, though many other Actinobacteria have both RqlH and RecQ. Whereas E. coli K12 encodes RecQ but lacks a homolog of RqlH, other strains of E. coli have both RqlH and RecQ.

Double strand break unwinding and resection by the mycobacterial helicase-nuclease AdnAB in the presence of single strand DNA-binding protein (SSB).

Mycobacterial AdnAB is a heterodimeric DNA helicase-nuclease and 3' to 5' DNA translocase implicated in the repair of double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Inclusion of mycobacterial single strand DNA-binding protein (SSB) in reactions containing linear plasmid dsDNA allowed us to study the AdnAB helicase under conditions in which the unwound single strands are coated by SSB and thereby prevented from reannealing or promoting ongoing ATP hydrolysis. We found that the AdnAB motor catalyzed processive unwinding of 2.7-11.2-kbp linear duplex DNAs at a rate of ∼250 bp s(-1), while hydrolyzing ∼5 ATPs per bp unwound. Crippling the AdnA phosphohydrolase active site did not affect the rate of unwinding but lowered energy consumption slightly, to ∼4.2 ATPs bp(-1). Mutation of the AdnB phosphohydrolase abolished duplex unwinding, consistent with a model in which the "leading" AdnB motor propagates a Y-fork by translocation along the 3' DNA strand, ahead of the "lagging" AdnA motor domain. By tracking the resection of the 5' and 3' strands at the DSB ends, we illuminated a division of labor among the AdnA and AdnB nuclease modules during dsDNA unwinding, whereby the AdnA nuclease processes the unwound 5' strand to liberate a short oligonucleotide product, and the AdnB nuclease incises the 3' strand on which the motor translocates. These results extend our understanding of presynaptic DSB processing by AdnAB and engender instructive comparisons with the RecBCD and AddAB clades of bacterial helicase-nuclease machines.

Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines.

Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

AdnAB: a new DSB-resecting motor-nuclease from mycobacteria.

The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase-nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase-nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16-17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor-nuclease complexes.

Domain requirements for DNA unwinding by mycobacterial UvrD2, an essential DNA helicase.

Mycobacterial UvrD2 is a DNA-dependent ATPase with 3' to 5' helicase activity. UvrD2 is an atypical helicase, insofar as its N-terminal ATPase domain resembles the superfamily I helicases UvrD/PcrA, yet it has a C-terminal HRDC domain, which is a feature of RecQ-type superfamily II helicases. The ATPase and HRDC domains are connected by a CxxC-(14)-CxxC tetracysteine module that defines a new clade of UvrD2-like bacterial helicases found only in Actinomycetales. By characterizing truncated versions of Mycobacterium smegmatis UvrD2, we show that whereas the HRDC domain is not required for ATPase or helicase activities in vitro, deletion of the tetracysteine module abolishes duplex unwinding while preserving ATP hydrolysis. Replacing each of the CxxC motifs with a double-alanine variant AxxA had no effect on duplex unwinding, signifying that the domain module, not the cysteines, is crucial for function. The helicase activity of a truncated UvrD2 lacking the tetracysteine and HRDC domains was restored by the DNA-binding protein Ku, a component of the mycobacterial NHEJ system and a cofactor for DNA unwinding by the paralogous mycobacterial helicase UvrD1. Our findings indicate that coupling of ATP hydrolysis to duplex unwinding can be achieved by protein domains acting in cis or trans. Attempts to disrupt the M. smegmatis uvrD2 gene were unsuccessful unless a second copy of uvrD2 was present elsewhere in the chromosome, indicating that UvrD2 is essential for growth of M. smegmatis.

In vivo iron-sulfur cluster formation.

It has been proposed that iron-sulfur [Fe-S] clusters destined for the maturation of [Fe-S] proteins can be preassembled on a molecular scaffold designated IscU. In the present article, it is shown that production of the intact Azotobacter vinelandii [Fe-S] cluster biosynthetic machinery at levels exceeding the amount required for cellular maturation of [Fe-S] proteins results in the accumulation of: (i) apo-IscU, (ii) an oxygen-labile [2Fe-2S] cluster-loaded form of IscU, and (iii) IscU complexed with the S-delivery protein, IscS. It is suggested that these species represent different stages of the [Fe-S] cluster assembly process. Substitution of the IscU Asp(39) residue by Ala results in the in vivo trapping of a stoichiometric, noncovalent, nondissociating IscU-IscS complex that contains an oxygen-resistant [Fe-S] species. In aggregate, these results validate the scaffold hypothesis for [Fe-S] cluster assembly and indicate that in vivo [Fe-S] cluster formation is a dynamic process that involves the reversible interaction of IscU and IscS.

Inhibitors of the molybdenum cofactor containing 4-hydroxybenzoyl-CoA reductase.

4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a member of the xanthine oxidase (XO) family of molybdenum cofactor containing enzymes and catalyzes the irreversible removal of a phenolic hydroxy group by reduction, yielding benzoyl-CoA and water. In this work the effects of various activity modulating compounds were characterized by kinetic, electron paramagnetic resonance (EPR) spectroscopic, and X-ray crystallographic studies. 4-HBCR was readily inactivated by cyanide and by the reducing agents titanium(III) citrate and dithionite; in contrast, reduced viologens had no inhibitory effect. Cyanide inhibition occurred in both the oxidized and reduced state of 4-HBCR. In the reduced state, cyanide-inhibited 4-HBCR was reactivated by simple oxidation. In contrast, reactivation from the oxidized state was only achieved in the presence of sulfide. Dithionite-inhibited 4-HBCR was reactivated by oxidation, whereas inhibition by titanium(III) citrate was irreversible. The previously reported inhibitory effect of azide could not be confirmed; instead, azide rather protected the enzyme from inactivation by titanium(III) citrate. The EPR spectra of the Mo(V) states were nearly identical in the noninhibited methyl viologen and in the dithionite-inhibited states of 4-HBCR; they exhibited a hyperfine splitting due to magnetic coupling with two solvent-exchangeable protons. The cyanide-treated enzyme showed the typical desulfo-inhibited Mo(V) EPR signal in D 2O, whereas in H 2O the hyperfine splitting was altered but indicated no loss of Mo(V)-proton interactions. The structures of dithionite- and azide-bound 4-HBCR were solved at 2.1 and 2.2 A, respectively. Both dithionite and azide bound directly to equatorial ligation sites of the Mo atom. The results obtained revealed further insights into the active site of an unusual member of the XO family of molybdenum cofactor containing enzymes.