Effects of DNA3'pp5'G capping on 3' end repair reactions and of an embedded pyrophosphate-linked guanylate on ribonucleotide surveillance.

When DNA breakage results in a 3'-PO4 terminus, the end is considered 'dirty' because it cannot prime repair synthesis by DNA polymerases or sealing by classic DNA ligases. The noncanonical ligase RtcB can guanylylate the DNA 3'-PO4 to form a DNA3'pp5'GOH cap. Here we show that DNA capping precludes end joining by classic ATP-dependent and NAD(+)-dependent DNA ligases, prevents template-independent nucleotide addition by mammalian terminal transferase, blocks exonucleolytic proofreading by Escherichia coli DNA polymerase II and inhibits proofreading by E. coli DNA polymerase III, while permitting templated DNA synthesis from the cap guanosine 3'-OH primer by E. coli DNA polymerase II (B family) and E. coli DNA polymerase III (C family). Human DNA polymerase ß (X family) extends the cap primer predominantly by a single templated addition step. Cap-primed synthesis by templated polymerases embeds a pyrophosphate-linked ribonucleotide in DNA. We find that the embedded ppG is refractory to surveillance and incision by RNase H2.

Impact of DNA3'pp5'G capping on repair reactions at DNA 3' ends.

Many biological scenarios generate "dirty" DNA 3'-PO4 ends that cannot be sealed by classic DNA ligases or extended by DNA polymerases. The noncanonical ligase RtcB can "cap" these ends via a unique chemical mechanism entailing transfer of GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form DNA3'pp5'G. Here, we show that capping protects DNA 3' ends from resection by Escherichia coli exonucleases I and III and from end-healing by T4 polynucleotide 3' phosphatase. By contrast, the cap is an effective primer for DNA synthesis. E. coli DNA polymerase I and Mycobacterium DinB1 extend the DNAppG primer to form an alkali-labile DNApp(rG)pDNA product. The addition of dNTP depends on pairing of the cap guanine with an opposing cytosine in the template strand. Aprataxin, an enzyme implicated in repair of A5'pp5'DNA ends formed during abortive ligation by classic ligases, is highly effective as a DNA 3' decapping enzyme, converting DNAppG to DNA3'p and GMP. We conclude that the biochemical impact of DNA capping is to prevent resection and healing of a 3'-PO4 end, while permitting DNA synthesis, at the price of embedding a ribonucleotide and a pyrophosphate linkage in the repaired strand. Aprataxin affords a means to counter the impact of DNA capping.

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP•Mg2+ and 5'-OH oligonucleotide and a product complex with GDP•Mg2+ and 5'-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition.

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5' end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5'-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg(2+) and a 5'-OH oligonucleotide and a product complex with GDP•Mg(2+) and a 5'-PO4 oligonucleotide. The O5' nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging ß phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5'-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38-hereby implicated as the essential general base catalyst that abstracts a proton from the 5'-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the 'reverse kinase' reaction by donating a proton to the O5' leaving group of the 5'-PO4 strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.

Rewriting the rules for end joining via enzymatic splicing of DNA 3'-PO4 and 5'-OH ends.

There are many biological contexts in which DNA damage generates "dirty" breaks with 3'-PO4 (or cyclic-PO4) and 5'-OH ends that cannot be sealed by DNA ligases. Here we show that the Escherichia coli RNA ligase RtcB can splice these dirty DNA ends via a unique chemical mechanism. RtcB transfers GMP from a covalent RtcB-GMP intermediate to a DNA 3'-PO4 to form a "capped" 3' end structure, DNA3'pp5'G. When a suitable DNA 5'-OH end is available, RtcB catalyzes attack of the 5'-OH on DNA3'pp5'G to form a 3'-5' phosphodiester splice junction. Our findings unveil an enzymatic capacity for DNA 3' capping and the sealing of DNA breaks with 3'-PO4 and 5'-OH termini, with implications for DNA repair and DNA rearrangements.

2'-Phosphate cyclase activity of RtcA: a potential rationale for the operon organization of RtcA with an RNA repair ligase RtcB in Escherichia coli and other bacterial taxa.

RNA terminal phosphate cyclase catalyzes the ATP-dependent conversion of a 3'-phosphate RNA end to a 2',3'-cyclic phosphate via covalent enzyme-(histidinyl-Nε)-AMP and RNA(3')pp(5')A intermediates. Here, we report that Escherichia coli RtcA (and its human homolog Rtc1) are capable of cyclizing a 2'-phosphate RNA end in high yield. The rate of 2'-phosphate cyclization by RtcA is five orders of magnitude slower than 3'-phosphate cyclization, notwithstanding that RtcA binds with similar affinity to RNA3'p and RNA2'p substrates. These findings expand the functional repertoire of RNA cyclase and suggest that phosphate geometry during adenylate transfer to RNA is a major factor in the kinetics of cyclization. RtcA is coregulated in an operon with an RNA ligase, RtcB, that splices RNA 5'-OH ends to either 3'-phosphate or 2',3'-cyclic phosphate ends. Our results suggest that RtcA might serve an end healing function in an RNA repair pathway, by converting RNA 2'-phosphates, which cannot be spliced by RtcB, to 2',3'-cyclic phosphates that can be sealed. The rtcBA operon is controlled by the σ(54) coactivator RtcR encoded by an adjacent gene. This operon arrangement is conserved in diverse bacterial taxa, many of which have also incorporated the RNA-binding protein Ro (which is implicated in RNA quality control under stress conditions) as a coregulated component of the operon.

Mechanism of RNA 2',3'-cyclic phosphate end healing by T4 polynucleotide kinase-phosphatase.

T4 polynucleotide kinase-phosphatase (Pnkp) exemplifies a family of enzymes with 5'-kinase and 3'-phosphatase activities that function in nucleic acid repair. The polynucleotide 3'-phosphatase reaction is executed by the Pnkp C-terminal domain, which belongs to the DxDxT acylphosphatase superfamily. The 3'-phosphatase reaction entails formation and hydrolysis of a covalent enzyme-(Asp165)-phosphate intermediate, driven by general acid-base catalyst Asp167. We report that Pnkp also has RNA 2'-phosphatase activity that requires Asp165 and Asp167. The physiological substrate for Pnkp phosphatase is an RNA 2',3'-cyclic phosphate end (RNA > p), but the pathway of cyclic phosphate removal and its enzymic requirements are undefined. Here we find that Pnkp reactivity with RNA > p requires Asp165, but not Asp167. Whereas wild-type Pnkp transforms RNA > p to RNA(OH), mutant D167N converts RNA > p to RNA 3'-phosphate, which it sequesters in the phosphatase active site. In support of the intermediacy of an RNA phosphomonoester, the reaction of mutant S211A with RNA > p results in transient accumulation of RNAp en route to RNA(OH). Our results suggest that healing of 2',3'-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3'-phosphoaspartyl)-Pnkp → RNA(3')p + Pnkp → RNA(OH) + phosphoaspartyl-Pnkp → P(i) + Pnkp.

Structures of bacterial polynucleotide kinase in a michaelis complex with nucleoside triphosphate (NTP)-Mg2+ and 5'-OH RNA and a mixed substrate-product complex with NTP-Mg2+ and a 5'-phosphorylated oligonucleotide.

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5'-end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from a nucleoside triphosphate (NTP) donor to a 5'-OH polynucleotide acceptor, either DNA or RNA. Here we report the 1.5-Šcrystal structure of CthPnk-D38N in a Michaelis complex with GTP-Mg(2+) and a 5'-OH RNA oligonucleotide. The RNA-binding mode of CthPnk is different from that of the metazoan RNA kinase Clp1. CthPnk makes hydrogen bonds to the ribose 2'-hydroxyls of the 5' terminal nucleoside, via Gln51, and the penultimate nucleoside, via Gln83. The 5'-terminal nucleobase is sandwiched by Gln51 and Val129. Mutating Gln51 or Val129 to alanine reduced kinase specific activity 3-fold. Ser37 and Thr80 donate functionally redundant hydrogen bonds to the terminal phosphodiester; a S37A-T80A double mutation reduced kinase activity 50-fold. Crystallization of catalytically active CthPnk with GTP-Mg(2+) and a 5'-OH DNA yielded a mixed substrate-product complex with GTP-Mg(2+) and 5'-PO4 DNA, wherein the product 5' phosphate group is displaced by the NTP γ phosphate and the local architecture of the acceptor site is perturbed.

Structure and mechanism of the polynucleotide kinase component of the bacterial Pnkp-Hen1 RNA repair system.

Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5'-kinase, a central 2',3' phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATP•Mg²âº (substrate complex) and ADP•Mg²âº (product complex). The protein consists of a core P-loop phosphotransferase fold embellished by a distinctive homodimerization module composed of secondary structure elements derived from the N and C termini of the kinase domain. ATP is bound within a crescent-shaped groove formed by the P-loop (¹5GSSGSGKST²³) and an overlying helix-loop-helix "lid." The α and ß phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the γ phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg²âº bridge the ATP ß and γ phosphates. The P-loop serine (Ser22) is the sole enzymic constituent of the octahedral metal coordination complex. Structure-guided mutational analysis underscored the essential contributions of Lys21 and Ser22 in the ATP donor site and Asp38 and Arg41 in the phosphoacceptor site. Our studies suggest a catalytic mechanism whereby Asp38 (as general base) activates the polynucleotide 5'-OH for its nucleophilic attack on the γ phosphorus and Lys21 and Mg²âº stabilize the transition state.

Structural insights to the metal specificity of an archaeal member of the LigD 3'-phosphoesterase DNA repair enzyme family.

LigD 3'-phosphoesterase (PE) enzymes perform end-healing reactions at DNA breaks. Here we characterize the 3'-ribonucleoside-resecting activity of Candidatus Korarchaeum PE. CkoPE prefers a single-stranded substrate versus a primer-template. Activity is abolished by vanadate (10 mM), but is less sensitive to phosphate (IC(50) 50 mM) or chloride (IC(50) 150 mM). The metal requirement is satisfied by manganese, cobalt, copper or cadmium, but not magnesium, calcium, nickel or zinc. Insights to CkoPE metal specificity were gained by solving new 1.5 Å crystal structures of CkoPE in complexes with Co(2+) and Zn(2+). His9, His15 and Asp17 coordinate cobalt in an octahedral complex that includes a phosphate anion, which is in turn coordinated by Arg19 and His51. The cobalt and phosphate positions and the atomic contacts in the active site are virtually identical to those in the CkoPE·Mn(2+) structure. By contrast, Zn(2+) binds in the active site in a tetrahedral complex, wherein the position, orientation and atomic contacts of the phosphate are shifted and its interaction with His51 is lost. We conclude that: (i) PE selectively binds to 'soft' metals in either productive or non-productive modes and (ii) PE catalysis depends acutely on proper metal and scissile phosphate geometry.

Structural and biochemical analysis of the phosphate donor specificity of the polynucleotide kinase component of the bacterial pnkp•hen1 RNA repair system.

Clostridium thermocellum Pnkp is the end-healing and end-sealing subunit of a bacterial RNA repair system. CthPnkp is composed of three catalytic modules: an N-terminal 5'-OH polynucleotide kinase, a central 2',3' phosphatase, and a C-terminal ligase. The crystal structure of the kinase domain bound to ATP•Mg(2+) revealed a rich network of ionic and hydrogen-bonding contacts to the α, ß, and γ phosphates. By contrast, there are no enzymic contacts to the ribose and none with the adenine base other than a π-cation interaction with Arg116. Here we report that the enzyme uses ATP, GTP, CTP, UTP, or dATP as a phosphate donor for the 5'-OH kinase reaction. The enzyme also catalyzes the reverse reaction, in which a polynucleotide 5'-PO4 group is transferred to ADP, GDP, CDP, UDP, or dADP to form the corresponding NTP. We report new crystal structures of the kinase in complexes with GTP, CTP, UTP, and dATP in which the respective nucleobases are stacked on Arg116 but make no other enzymic contacts. Mutating Arg116 to alanine elicits a 10-fold increase in Km for ATP but has little effect on kcat. These findings illuminate the basis for nonspecific donor nucleotide utilization by a P-loop phosphotransferase.

Structures and activities of archaeal members of the LigD 3'-phosphoesterase DNA repair enzyme superfamily.

LigD 3'-phosphoesterase (PE) is a component of the bacterial NHEJ apparatus that performs 3'-end-healing reactions at DNA breaks. The tertiary structure, active site and substrate specificity of bacterial PE are unique vis-à-vis other end-healing enzymes. PE homologs are present in archaea, but their properties are uncharted. Here, we demonstrate the end-healing activities of two archaeal PEs--Candidatus Korarchaeum cryptofilum PE (CkoPE; 117 amino acids) and Methanosarcina barkeri PE (MbaPE; 151 amino acids)--and we report their atomic structures at 1.1 and 2.1 Å, respectively. Archaeal PEs are minimized versions of bacterial PE, consisting of an eight-stranded ß barrel and a 3(10) helix. Their active sites are located in a crescent-shaped groove on the barrel's outer surface, wherein two histidines and an aspartate coordinate manganese in an octahedral complex that includes two waters and a phosphate anion. The phosphate is in turn coordinated by arginine and histidine side chains. The conservation of active site architecture in bacterial and archaeal PEs, and the concordant effects of active site mutations, underscore a common catalytic mechanism, entailing transition state stabilization by manganese and the phosphate-binding arginine and histidine. Our results fortify the proposal that PEs comprise a DNA repair superfamily distributed widely among taxa.

N-urethane-protected amino alkyl isothiocyanates: synthesis, isolation, characterization, and application to the synthesis of thioureidopeptides.

Synthetically useful N-Fmoc amino-alkyl isothiocyanates have been described, starting from protected amino acids. These compounds have been synthesized in excellent yields by thiocarbonylation of the monoprotected 1,2-diamines with CS2/TEA/p-TsCl, isolated as stable solids, and completely characterized. The procedure has been extended to the synthesis of amino alkyl isothiocyanates from Boc- and Z-protected amino acids as well. The utility of these isothiocyanates for peptidomimetics synthesis has been demonstrated by employing them in the preparation of a series of dithioureidopeptide esters. Boc-Gly-OH and Boc-Phe-OH derived isothiocyanates 9a and 9c have been obtained as single crystals and their structures solved through X-ray diffraction. They belong to the orthorhombic crystal system, and have a single molecule in the asymmetric unit (Z' = 1). 9a crystallizes in the centrosymmetric space group Pbca, while 9c crystallizes in the noncentrosymmetric space group P2(1)2(1)2(1).

4-(4-Methoxy-phen-yl)-1-phenyl-pyridine-2,6(1H,3H)-dione.

In the title compound, C(18)H(15)NO(3), the pyridine-2,6-dione ring adopts an envelope conformation. The phenyl ring lies approximately perpendicular to the mean plane of the pyridine-2,6-dione ring [dihedral angle = 81.5 (1)°], while the methoxy-phenyl ring is tilted to the same plane by a dihedral angle of 34.8 (1)°. Inter-molecular C-H⋯O inter-actions link the mol-ecules into chains along [100].

3-(4-Methoxy-phen-yl)pent-2-ene-1,5-dioic acid.

In the title compound, C(12)H(12)O(5), mol-ecules are linked into anti-parallel hydrogen-bonded sheets through inversion dimers generated via two O-H⋯O hydrogen bonds. Using the R(2) (2)(8) motif as a building block, hydrogen-bonded chains of a C(2) (2)(8) superstructure are then generated.

Ethyl 4-(4-hydroxy-phen-yl)-6-methyl-2-oxo-1,2,3,4-tetra-hydro-pyrimidine-5-carboxyl-ate monohydrate.

There are three formula units in the asymmetric unit of the title compound, C(14)H(16)N(2)O(4)·H(2)O. Mol-ecules are linked by N-H⋯O hydrogen bonds into dimers with the common R(2) (2)(8) graph-set motif. Between dimers, single N-H⋯O hydrogen bonds are formed between the other N-H group of each pyrimidine ring and the hydroxyl groups. The water mol-ecules accept O-H⋯O hydrogen bonds from the hydroxyl groups and donate hydrogen bonds to the ester groups.

Engineering of a miniaturized, robotic clinical laboratory.

The ability to perform laboratory testing near the patient and with smaller blood volumes would benefit patients and physicians alike. We describe our design of a miniaturized clinical laboratory system with three components: a hardware platform (ie, the miniLab) that performs preanalytical and analytical processing steps using miniaturized sample manipulation and detection modules, an assay-configurable cartridge that provides consumable materials and assay reagents, and a server that communicates bidirectionally with the miniLab to manage assay-specific protocols and analyze, store, and report results (i.e., the virtual analyzer). The miniLab can detect analytes in blood using multiple methods, including molecular diagnostics, immunoassays, clinical chemistry, and hematology. Analytical performance results show that our qualitative Zika virus assay has a limit of detection of 55 genomic copies/ml. For our anti-herpes simplex virus type 2 immunoglobulin G, lipid panel, and lymphocyte subset panel assays, the miniLab has low imprecision, and method comparison results agree well with those from the United States Food and Drug Administration-cleared devices. With its small footprint and versatility, the miniLab has the potential to provide testing of a range of analytes in decentralized locations.