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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 59-kinase, a central 29,39 phosphatase, and a C-terminal ligase. Here we report the crystal structure of the kinase domain of Clostridium thermocellum Pnkp bound to ATPdMg 2+ (substrate complex) and ADPdMg 2+ (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 ( 15 GSSGSGKST 23 ) and an overlying helix-loop-helix ''lid.'' The a and b phosphates are engaged by a network of hydrogen bonds from Thr23 and the P-loop main-chain amides; the g phosphate is anchored by the lid residues Arg120 and Arg123. The P-loop lysine (Lys21) and the catalytic Mg 2+ bridge the ATP b and g 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 59-OH for its nucleophilic attack on the g phosphorus and Lys21 and Mg 2+ stabilize the transition state.

There are many biological contexts in which DNA damage generates "dirty" breaks with 3′-PO 4 (or cyclic-PO 4 ) 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′-PO 4 to form a "capped" 3′ end structure, DNA 3′ pp 5′ G. When a suitable DNA 5′-OH end is available, RtcB catalyzes attack of the 5′-OH on DNA 3′ pp 5′ 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′-PO 4 and 5′-OH termini, with implications for DNA repair and DNA rearrangements.T he Escherichia coli RtcB is a founding member of a recently discovered family of RNA repair/splicing enzymes that join RNA 2′,3′-cyclic-PO 4 or 3′-PO 4 ends to RNA 5′-OH ends (1-4). RtcB executes a four-step pathway that requires GTP as an energy source and Mn 2+ as a cofactor (5-7). RtcB first reacts with GTP to form a covalent RtcB-(histidinyl 337 -N)-GMP intermediate. It then hydrolyzes the RNA 2′,3′-cyclic-PO 4 end to a 3′-PO 4 and transfers guanylate from His337 to the RNA 3′-PO 4 to form an RNA 3′ pp 5′ G intermediate. Finally, RtcB catalyzes the attack of an RNA 5′-OH on the RNA 3′ pp 5′ G end to form the 3′-5′ phosphodiester splice junction and liberate GMP.The unique chemical mechanism of RtcB overturned a longstanding tenet of nucleic acid enzymology, which held that synthesis of polynucleotide 3′-5′ phosphodiesters proceeds via the attack of a 3′-OH on a high-energy 5′-phosphoanhydride: either a nucleoside 5′-triphosphate in the case of RNA/DNA polymerases or an adenylylated intermediate A 5′ pp 5′ N, in the case of classic RNA/DNA ligases. In light of the wide distribution of RtcB proteins in bacteria, archaea, and metazoa, we raised the prospect of an alternative enzymology based on covalently activated 3′-PO 4 ends (6).In principle, the chemistry of RNA 3′-PO 4 /5′-OH end joining by RtcB might be portable to DNA transactions and pertinent to DNA repair. A variety of hydrolytic nucleases incise the DNA phosphodiester backbone to yield 3′-PO 4 and 5′-OH termini that cannot be joined by DNA ligases. Nonligatable 3′-PO 4 ends are also generated during base excision repair catalyzed by DNA glycosylase/lyase enzymes, during the repair of trapped covalent topoisomerase IB-DNA adducts by tyrosyl-DNA phosphodiesterase 1, and during DNA damage inflicted by ionizing radiation. One way nature solves this "dirty end" problem is by deploying a variety of "end healing" enzymes (8-14). These include 3′-phosphoesterases that convert a 3′-PO 4 to a 3′-OH and 5′-kinases that transform a 5′-OH to a 5′-PO 4 , thereby enabling break sealing by the classic ligase pathway. Given what we now know about RtcB, would it not make sense for nature to also endow a pathway for direct joining of DNA 3′-PO 4 and 5′-OH ends, be it via RtcB or another ligase yet to be discovered?We can extend this thought to DN...

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 RNAOH, 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 RNAOH. Our results suggest that healing of 2′,3′-cyclic phosphate ends is a four-step processive reaction: RNA > p + Pnkp → RNA-(3′-phosphoaspartyl)-Pnkp → RNA3′p + Pnkp → RNAOH + phosphoaspartyl-Pnkp → Pi + Pnkp.

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).