RNA Repair: Hiding in Plain Sight.

Enzymes that phosphorylate, dephosphorylate, and ligate RNA 5' and 3' ends were discovered more than half a century ago and were eventually shown to repair purposeful site-specific endonucleolytic breaks in the RNA phosphodiester backbone. The pace of discovery and characterization of new candidate RNA repair activities in taxa from all phylogenetic domains greatly exceeds our understanding of the biological pathways in which they act. The key questions anent RNA break repair in vivo are (a) identifying the triggers, agents, and targets of RNA cleavage and (b) determining whether RNA repair results in restoration of the original RNA, modification of the RNA (by loss or gain at the ends), or rearrangements of the broken RNA segments (i.e., RNA recombination). This review provides a perspective on the discovery, mechanisms, and physiology of purposeful RNA break repair, highlighting exemplary repair pathways (e.g., tRNA restriction-repair and tRNA splicing) for which genetics has figured prominently in their elucidation.

Structure-function analysis of STING activation by c[G(2',5')pA(3',5')p] and targeting by antiviral DMXAA.

Binding of dsDNA by cyclic GMP-AMP (cGAMP) synthase (cGAS) triggers formation of the metazoan second messenger c[G(2',5')pA(3',5')p], which binds the signaling protein STING with subsequent activation of the interferon (IFN) pathway. We show that human hSTING(H232) adopts a "closed" conformation upon binding c[G(2',5')pA(3',5')p] and its linkage isomer c[G(2',5')pA(2',5')p], as does mouse mSting(R231) on binding c[G(2',5')pA(3',5')p], c[G(3',5')pA(3',5')p] and the antiviral agent DMXAA, leading to similar "closed" conformations. Comparing hSTING to mSting, 2',5'-linkage-containing cGAMP isomers were more specific triggers of the IFN pathway compared to the all-3',5'-linkage isomer. Guided by structural information, we identified a unique point mutation (S162A) placed within the cyclic-dinucleotide-binding site of hSTING that rendered it sensitive to the otherwise mouse-specific drug DMXAA, a conclusion validated by binding studies. Our structural and functional analysis highlights the unexpected versatility of STING in the recognition of natural and synthetic ligands within a small-molecule pocket created by the dimerization of STING.

Kinetic and structural insights into the requirement of fungal tRNA ligase for a 2'-phosphate end.

Fungal RNA ligase (LIG) is an essential tRNA splicing enzyme that joins 3'-OH,2'-PO4 and 5'-PO4 RNA ends to form a 2'-PO4,3'-5' phosphodiester splice junction. Sealing entails three divalent cation-dependent adenylate transfer steps. First, LIG reacts with ATP to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and displace pyrophosphate. Second, LIG transfers AMP to the 5'-PO4 RNA terminus to form an RNA-adenylate intermediate (A5'pp5'RNA). Third, LIG directs the attack of an RNA 3'-OH on AppRNA to form the splice junction and displace AMP. A defining feature of fungal LIG vis-à-vis canonical polynucleotide ligases is the requirement for a 2'-PO4 to synthesize a 3'-5' phosphodiester bond. Fungal LIG consists of an N-terminal adenylyltransferase domain and a unique C-terminal domain. The C-domain of Chaetomium thermophilum LIG (CthLIG) engages a sulfate anion thought to be a mimetic of the terminal 2'-PO4. Here were interrogated the contributions of the C-domain and the conserved sulfate ligands (His227, Arg334, Arg337) to ligation of a pRNA2'p substrate. We find that the C-domain is essential for end-joining but dispensable for ligase adenylylation. Mutations H227A, R334A, and R337A slowed the rate of step 2 RNA adenylation by 420-fold, 120-fold, and 60-fold, respectively, vis-à-vis wild-type CthLIG. An R334A-R337A double-mutation slowed step 2 by 580-fold. These results fortify the case for the strictly conserved His-Arg-Arg triad as the enforcer of the 2'-PO4 end-specificity of fungal tRNA ligases and as a target for small molecule interdiction of fungal tRNA splicing.

Characterization of tRNA splicing enzymes RNA ligase and tRNA 2'-phosphotransferase from the pathogenic fungi Mucorales.

Fungal Trl1 is an essential trifunctional tRNA splicing enzyme that heals and seals tRNA exons with 2',3'-cyclic-PO4 and 5'-OH ends. Trl1 is composed of C-terminal cyclic phosphodiesterase and central polynucleotide kinase end-healing domains that generate the 3'-OH,2'-PO4 and 5'-PO4 termini required for sealing by an N-terminal ATP-dependent ligase domain. Trl1 enzymes are present in many human fungal pathogens and are promising targets for antifungal drug discovery because their domain structures and biochemical mechanisms are unique compared to the mammalian RtcB-type tRNA splicing enzyme. Here we report that Mucorales species (deemed high-priority human pathogens by WHO) elaborate a noncanonical tRNA splicing apparatus in which a monofunctional RNA ligase enzyme is encoded separately from any end-healing enzymes. We show that Mucor circinelloides RNA ligase (MciRNL) is active in tRNA splicing in vivo in budding yeast in lieu of the Trl1 ligase domain. Biochemical and kinetic characterization of recombinant MciRNL underscores its requirement for a 2'-PO4 terminus in the end-joining reaction, whereby the 2'-PO4 enhances the rates of RNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3) by ∼125-fold and ∼6200-fold, respectively. In the canonical fungal tRNA splicing pathway, the splice junction 2'-PO4 installed by RNA ligase is removed by a dedicated NAD+-dependent RNA 2'-phosphotransferase Tpt1. Here we identify and affirm by genetic complementation in yeast the biological activity of Tpt1 orthologs from three Mucorales species. Recombinant M. circinelloides Tpt1 has vigorous NAD+-dependent RNA 2'-phosphotransferase activity in vitro.

Structural basis for Tpt1-catalyzed 2'-PO4 transfer from RNA and NADP(H) to NAD.

Tpt1 is an essential agent of fungal and plant tRNA splicing that removes an internal RNA 2'-phosphate generated by tRNA ligase. Tpt1 also removes the 2'-phosphouridine mark installed by Ark1 kinase in the V-loop of archaeal tRNAs. Tpt1 performs a two-step reaction in which the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate, and transesterification of the ADP-ribose O2″ to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate. Here, we present structures of archaeal Tpt1 enzymes, captured as product complexes with ADP-ribose-1″-PO4, ADP-ribose-2″-PO4, and 2'-OH RNA, and as substrate complexes with 2',5'-ADP and NAD+, that illuminate 2'-PO4 junction recognition and catalysis. We show that archaeal Tpt1 enzymes can use the 2'-PO4-containing metabolites NADP+ and NADPH as substrates for 2'-PO4 transfer to NAD+. A role in 2'-phospho-NADP(H) dynamics provides a rationale for the prevalence of Tpt1 in taxa that lack a capacity for internal RNA 2'-phosphorylation.

Factors governing the transcriptome changes and chronological lifespan of fission yeast during phosphate starvation.

Starvation of Schizosaccharomyces pombe for inorganic phosphate elicits adaptive transcriptome changes in which mRNAs driving ribosome biogenesis, tRNA biogenesis, and translation are globally downregulated, while those for autophagy and phosphate mobilization are upregulated. Here, we interrogated three components of the starvation response: upregulated autophagy; the role of transcription factor Pho7 (an activator of the PHO regulon); and upregulated expression of ecl3, one of three paralogous genes (ecl1, ecl2, and ecl3) collectively implicated in cell survival during other nutrient stresses. Ablation of autophagy factor Atg1 resulted in early demise of phosphate-starved fission yeast, as did ablation of Pho7. Transcriptome profiling of phosphate-starved pho7Δ cells highlighted Pho7 as an activator of genes involved in phosphate acquisition and mobilization, not limited to the original three-gene PHO regulon, and additional starvation-induced genes (including ecl3) not connected to phosphate dynamics. Pho7-dependent gene induction during phosphate starvation tracked with the presence of Pho7 DNA-binding elements in the gene promoter regions. Fewer ribosome protein genes were downregulated in phosphate-starved pho7Δ cells versus WT, which might contribute to their shortened lifespan. An ecl3Δ mutant elicited no gene expression changes in phosphate-replete cells and had no impact on survival during phosphate starvation. By contrast, pan-ecl deletion (ecl123Δ) curtailed lifespan during chronic phosphate starvation. Phosphate-starved ecl123Δ cells experienced a more widespread downregulation of mRNAs encoding aminoacyl tRNA synthetases vis-à-vis WT or pho7Δ cells. Collectively, these results enhance our understanding of fission yeast phosphate homeostasis and survival during nutrient deprivation.

Fission yeast poly(A) polymerase active site mutation Y86D alleviates the rad24Δ asp1-H397A synthetic growth defect and up-regulates mRNAs targeted by MTREC and Mmi1.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA-mediated interference is alleviated by genetic perturbations that elicit precocious lncRNA 3'-processing and transcription termination, such as (i) the inositol pyrophosphate pyrophosphatase-defective asp1-H397A allele, which results in elevated levels of IP8, and (ii) absence of the 14-3-3 protein Rad24. Combining rad24Δ with asp1-H397A causes a severe synthetic growth defect. A forward genetic screen for SRA (Suppressor of Rad24 Asp1-H397A) mutations identified a novel missense mutation (Tyr86Asp) of Pla1, the essential poly(A) polymerase subunit of the fission yeast cleavage and polyadenylation factor (CPF) complex. The pla1-Y86D allele was viable but slow-growing in an otherwise wild-type background. Tyr86 is a conserved active site constituent that contacts the RNA primer 3' nt and the incoming ATP. The Y86D mutation elicits a severe catalytic defect in RNA-primed poly(A) synthesis in vitro and in binding to an RNA primer. Yet, analyses of specific mRNAs indicate that poly(A) tails in pla1-Y86D cells are not different in size than those in wild-type cells, suggesting that other RNA interactors within CPF compensate for the defects of isolated Pla1-Y86D. Transcriptome profiling of pla1-Y86D cells revealed the accumulation of multiple RNAs that are normally rapidly degraded by the nuclear exosome under the direction of the MTREC complex, with which Pla1 associates. We suggest that Pla1-Y86D is deficient in the hyperadenylation of MTREC targets that precedes their decay by the exosome.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Cellular strategies for regulating DNA supercoiling: a single-molecule perspective.

Entangling and twisting of cellular DNA (i.e., supercoiling) are problems inherent to the helical structure of double-stranded DNA. Supercoiling affects transcription, DNA replication, and chromosomal segregation. Consequently the cell must fine-tune supercoiling to optimize these key processes. Here, we summarize how supercoiling is generated and review experimental and theoretical insights into supercoil relaxation. We distinguish between the passive dissipation of supercoils by diffusion and the active removal of supercoils by topoisomerase enzymes. We also review single-molecule studies that elucidate the timescales and mechanisms of supercoil removal.

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.

Cellular responses to long-term phosphate starvation of fission yeast: Maf1 determines fate choice between quiescence and death associated with aberrant tRNA biogenesis.

Inorganic phosphate is an essential nutrient acquired by cells from their environment. Here, we characterize the adaptative responses of fission yeast to chronic phosphate starvation, during which cells enter a state of quiescence, initially fully reversible upon replenishing phosphate after 2 days but resulting in gradual loss of viability during 4 weeks of starvation. Time-resolved analyses of changes in mRNA levels revealed a coherent transcriptional program in which phosphate dynamics and autophagy were upregulated, while the machineries for rRNA synthesis and ribosome assembly, and for tRNA synthesis and maturation, were downregulated in tandem with global repression of genes encoding ribosomal proteins and translation factors. Consistent with the transcriptome changes, proteome analysis highlighted global depletion of 102 ribosomal proteins. Concomitant with this ribosomal protein deficit, 28S and 18S rRNAs became vulnerable to site-specific cleavages that generated temporally stable rRNA fragments. The finding that Maf1, a repressor of RNA polymerase III transcription, was upregulated during phosphate starvation prompted a hypothesis that its activity might prolong lifespan of the quiescent cells by limiting production of tRNAs. Indeed, we found that deletion of maf1 results in precocious death of phosphate-starved cells via a distinctive starvation-induced pathway associated with tRNA overproduction and dysfunctional tRNA biogenesis.

Structures of RNA ligase RtcB in complexes with divalent cations and GTP.

Pyrococcus horikoshii (Pho) RtcB exemplifies a family of binuclear transition metal- and GTP-dependent RNA ligases that join 3'-phosphate and 5'-OH ends via RtcB-(histidinyl-N)-GMP and RNA3'pp5'G intermediates. We find that guanylylation of PhoRtcB is optimal with manganese and less effective with cobalt and nickel. Zinc and copper are inactive and potently inhibit manganese-dependent guanylylation. We report crystal structures of PhoRtcB in complexes with GTP and permissive (Mn, Co, Ni) or inhibitory (Zn, Cu) metals. Zinc and copper occupy the M1 and M2 sites adjacent to the GTP phosphates, as do manganese, cobalt, and nickel. The identity/positions of enzymic ligands for M1 (His234, His329, Cys98) and M2 (Cys98, Asp95, His203) are the same for permissive and inhibitory metals. The differences pertain to: (i) the coordination geometries and phosphate contacts of the metals; and (ii) the orientation of the His404 nucleophile with respect to the GTP α-phosphate and pyrophosphate leaving group. M2 metal coordination geometry correlates with metal cofactor activity, whereby inhibitory Zn2 and Cu2 assume a tetrahedral configuration and contact only the GTP γ-phosphate, whereas Mn2, Co2, and Ni2 coordination complexes are pentahedral and contact the ß- and γ-phosphates. The His404-Nε-Pα-O(α-ß) angle is closer to apical in Mn (179°), Co (171°), and Ni (169°) structures than in Zn (160°) and Cu (155°) structures. The octahedral Mn1 geometry in our RtcB•GTP•Mn2+ structure, in which Mn1 contacts α-, ß-, and γ-phosphates, transitions to a tetrahedral configuration after formation of RtcB•(His404)-GMP•Mn2+ and departure of pyrophosphate.

Genetic screen for suppression of transcriptional interference reveals fission yeast 14-3-3 protein Rad24 as an antagonist of precocious Pol2 transcription termination.

Expression of fission yeast Pho1 acid phosphatase is repressed under phosphate-replete conditions by transcription of an upstream prt lncRNA that interferes with the pho1 mRNA promoter. lncRNA control of pho1 mRNA synthesis is influenced by inositol pyrophosphate (IPP) kinase Asp1, deletion of which results in pho1 hyper-repression. A forward genetic screen for ADS (Asp1 Deletion Suppressor) mutations identified the 14-3-3 protein Rad24 as a governor of phosphate homeostasis. Production of full-length interfering prt lncRNA was squelched in rad24Δ cells, concomitant with increased production of pho1 mRNA and increased Pho1 activity, while shorter precociously terminated non-interfering prt transcripts persisted. Epistasis analysis showed that pho1 de-repression by rad24Δ depends on: (i) 3'-processing and transcription termination factors CPF, Pin1, and Rhn1; and (ii) Threonine-4 of the Pol2 CTD. Combining rad24Δ with the IPP pyrophosphatase-dead asp1-H397A allele caused a severe synthetic growth defect that was ameliorated by loss-of-function mutations in CPF, Pin1, and Rhn1, and by CTD phospho-site mutations T4A and Y1F. Rad24 function in repressing pho1 was effaced by mutation of its phosphate-binding pocket. Our findings instate a new role for a 14-3-3 protein as an antagonist of precocious RNA 3'-processing/termination.

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.

Genetic screen for suppression of transcriptional interference identifies a gain-of-function mutation in Pol2 termination factor Seb1.

The system of long noncoding RNA (lncRNA)-mediated transcriptional interference that represses fission yeast phosphate homoeostasis gene pho1 provides a sensitive readout of genetic influences on cotranscriptional 3'-processing and termination and a tool for discovery of regulators of this phase of the Pol2 transcription cycle. Here, we conducted a genetic screen for relief of transcriptional interference that unveiled a mechanism by which Pol2 termination is enhanced via a gain-of-function mutation, G476S, in the RNA-binding domain of an essential termination factor, Seb1. The genetic and physical evidence for gain-of-function is compelling: 1) seb1-G476S de-represses pho1 and tgp1, both of which are subject to lncRNA-mediated transcriptional interference; 2) seb1-G476S elicits precocious lncRNA transcription termination in response to lncRNA 5'-proximal poly(A) signals; 3) seb1-G476S derepression of pho1 is effaced by loss-of-function mutations in cleavage and polyadenylation factor (CPF) subunits and termination factor Rhn1; 4) synthetic lethality of seb1-G476S with pho1 derepressive mutants rpb1-CTD-S7A and aps1∆ is rescued by CPF/Rhn1 loss-of-function alleles; and 5) seb1-G476S elicits an upstream shift in poly(A) site preference in several messenger RNA genes. A crystal structure of the Seb1-G476S RNA-binding domain indicates potential for gain of contacts from Ser476 to RNA nucleobases. To our knowledge, this is a unique instance of a gain-of-function phenotype in a eukaryal transcription termination protein.

Structure-function analysis of fission yeast cleavage and polyadenylation factor (CPF) subunit Ppn1 and its interactions with Dis2 and Swd22.

Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3' processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640-710. A TFIIS-like domain (aa 173-330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.

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.

Fission yeast Duf89 and Duf8901 are cobalt/nickel-dependent phosphatase-pyrophosphatases that act via a covalent aspartyl-phosphate intermediate.

Domain of Unknown Function 89 (DUF89) proteins are metal-dependent phosphohydrolases. Exemplary DUF89 enzymes differ in their metal and phosphosubstrate preferences. Here, we interrogated the activities and structures of two DUF89 paralogs from fission yeast-Duf89 and Duf8901. We find that Duf89 and Duf8901 are cobalt/nickel-dependent phosphohydrolases adept at hydrolyzing p-nitrophenylphosphate and PPi. Crystal structures of metal-free Duf89 and Co2+-bound Duf8901 disclosed two enzyme conformations that differed with respect to the position of a three-helix module, which is either oriented away from the active site in Duf89 or forms a lid over the active site in Duf8901. Lid closure results in a 16 Å movement of Duf8901 Asp195, vis-à-vis Asp199 in Duf89, that brings Asp195 into contact with an octahedrally coordinated cobalt. Reaction of Duf8901 with BeCl2 and NaF in the presence of divalent cations Co2+, Ni2+, or Zn2+ generated covalent Duf8901-(Asp248)-beryllium trifluoride (BeF3)•Co2+, Duf8901-(Asp248)-BeF3•Ni2+, or Duf8901-(Asp248)-BeF3•Zn2+ adducts, the structures of which suggest a two-step catalytic mechanism via formation and hydrolysis of an enzyme-(aspartyl)-phosphate intermediate. Alanine mutations of Duf8901 Asp248, Asn249, Lys401, Asp286, and Asp195 that interact with BeF3•Co2+ squelched p-nitrophenylphosphatase activity. A 1.8 Å structure of a Duf8901-(Asp248)-AlF4-OH2•Co2+ transition-state mimetic suggests an associative mechanism in which Asp195 and Asp363 orient and activate the water nucleophile. Whereas deletion of the duf89 gene elicited a phenotype in which expression of phosphate homeostasis gene pho1 was derepressed, deleting duf8901 did not, thereby hinting that the DUF89 paralogs have distinct functional repertoires in vivo.

Structure of 3'-PO4/5'-OH RNA ligase RtcB in complex with a 5'-OH oligonucleotide.

RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of Pyrococcus horikoshii RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide HOA1pT2pG3pT4pC5pC6, which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the A1pT2 and T2pG3 phosphates; Arg190 and Gln194 to the T2pG3 phosphate; and an Arg190 π-cation interaction with the G3 nucleobase. The structural insights affirm functional studies of E. coli RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.

Activity and substrate specificity of Candida, Aspergillus, and Coccidioides Tpt1: essential tRNA splicing enzymes and potential anti-fungal targets.

The enzyme Tpt1 is an essential agent of fungal tRNA splicing that removes an internal RNA 2'-PO4 generated by fungal tRNA ligase. Tpt1 performs a two-step reaction in which: (i) the 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-(ADP-ribose) intermediate; and (ii) transesterification of the ADP-ribose O2'' to the RNA 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1'',2''-cyclic phosphate. Because Tpt1 does not participate in metazoan tRNA splicing, and Tpt1 knockout has no apparent impact on mammalian physiology, Tpt1 is considered a potential anti-fungal drug target. Here we characterize Tpt1 enzymes from four human fungal pathogens: Coccidioides immitis, the agent of Valley Fever; Aspergillus fumigatus and Candida albicans, which cause invasive, often fatal, infections in immunocompromised hosts; and Candida auris, an emerging pathogen that is resistant to current therapies. All four pathogen Tpt1s were active in vivo in complementing a lethal Saccharomyces cerevisiae tpt1∆ mutation and in vitro in NAD+-dependent conversion of a 2'-PO4 to a 2'-OH. The fungal Tpt1s utilized nicotinamide hypoxanthine dinucleotide as a substrate in lieu of NAD+, albeit with much lower affinity, whereas nicotinic acid adenine dinucleotide was ineffective. Fungal Tpt1s efficiently removed an internal ribonucleotide 2'-phosphate from an otherwise all-DNA substrate. Replacement of an RNA ribose-2'-PO4 nucleotide with arabinose-2'-PO4 diminished enzyme specific activity by ≥2000-fold and selectively slowed step 2 of the reaction pathway, resulting in transient accumulation of an ara-2'-phospho-ADP-ribosylated intermediate. Our results implicate the 2'-PO4 ribonucleotide as the principal determinant of fungal Tpt1 nucleic acid substrate specificity.