Polynucleotide: adenosine glycosidase activity of saporin-L1: effect on DNA, RNA and poly(A).
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ABSTRACT:
SUBMITTER: Barbieri L
PROVIDER: S-EPMC1217797 | biostudies-other | 1996 Oct
REPOSITORIES: biostudies-other
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Project description:Ribosome inactivating proteins (RIPs) catalyze the hydrolytic depurination of one or more adenosine residues from eukaryotic ribosomes. Depurination of the ribosomal sarcin-ricin tetraloop (GAGA) causes inhibition of protein synthesis and cellular death. We characterized the catalytic properties of saporin-L1 from Saponaria officinalis (soapwort) leaves, and it demonstrated robust activity against defined nucleic acid substrates and mammalian ribosomes. Transition state analogue mimics of small oligonucleotide substrates of saporin-L1 are powerful, slow-onset inhibitors when adenosine is replaced with the transition state mimic 9-deazaadenine-9-methylene-N-hydroxypyrrolidine (DADMeA). Linear, cyclic, and stem-loop oligonucleotide inhibitors containing DADMeA and based on the GAGA sarcin-ricin tetraloop gave slow-onset tight-binding inhibition constants (K(i)*) of 2.3-8.7 nM under physiological conditions and bind up to 40000-fold tighter than RNA substrates. Saporin-L1 inhibition of rabbit reticulocyte translation was protected by these inhibitors. Transition state analogues of saporin-L1 have potential in cancer therapy that employs saporin-L1-linked immunotoxins.
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Project description:Saporin L3 from the leaves of the common soapwort is a catalyst for hydrolytic depurination of adenine from RNA. Saporin L3 is a type 1 ribosome inactivating protein (RIP) composed only of a catalytic domain. Other RIPs have been used in immunotoxin cancer therapy, but off-target effects have limited their development. In the current study, we use transition state theory to understand the chemical mechanism and transition state structure of saporin L3. In favorable cases, transition state structures guide the design of transition state analogues as inhibitors. Kinetic isotope effects (KIEs) were determined for an A14C mutant of saporin L3. To permit KIE measurements, small stem-loop RNAs that contain an AGGG tetraloop structure were enzymatically synthesized with the single adenylate bearing specific isotopic substitutions. KIEs were measured and corrected for forward commitment to obtain intrinsic values. A model of the transition state structure for depurination of stem-loop RNA (5'-GGGAGGGCCC-3') by saporin L3 was determined by matching KIE values predicted via quantum chemical calculations to a family of intrinsic KIEs. This model indicates saporin L3 displays a late transition state with the N-ribosidic bond to the adenine nearly cleaved, and the attacking water nucleophile weakly bonded to the ribosyl anomeric carbon. The transition state retains partial ribocation character, a feature common to most N-ribosyl transferases. However, the transition state geometry for saporin L3 is distinct from ricin A-chain, the only other RIP whose transition state is known.
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Project description:Phi29 DNA polymerase is a small DNA-dependent DNA polymerase that belongs to eukaryotic B-type DNA polymerases. Despite the small size, the polymerase is a multifunctional proofreading-proficient enzyme. It catalyzes two synthetic reactions (polymerization and deoxynucleotidylation of Phi29 terminal protein) and possesses two degradative activities (pyrophosphorolytic and 3'-->5' DNA exonucleolytic activities). Here we report that Phi29 DNA polymerase exonucleolyticaly degrades ssRNA. The RNase activity acts in a 3' to 5' polarity. Alanine replacements in conserved exonucleolytic site (D12A/D66A) inactivated RNase activity of the enzyme, suggesting that a single active site is responsible for cleavage of both substrates: DNA and RNA. However, the efficiency of RNA hydrolysis is approximately 10-fold lower than for DNA. Phi29 DNA polymerase is widely used in rolling circle amplification (RCA) experiments. We demonstrate that exoribonuclease activity of the enzyme can be used for the target RNA conversion into a primer for RCA, thus expanding application potential of this multifunctional enzyme and opening new opportunities for RNA detection.
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Project description:Saporin L3 from Saponaria officinalis (soapwort) leaves is a type 1 ribosome-inactivating protein. It catalyzes the hydrolysis of oligonucleotide adenylate N-ribosidic bonds to release adenine from rRNA. Depurination sites include both adenines in the GAGA tetraloop of short sarcin-ricin stem-loops and multiple adenines within eukaryotic rRNA, tRNAs, and mRNAs. Multiple Escherichia coli vector designs for saporin L3 expression were attempted but demonstrated high toxicity even during plasmid maintenance and selection in E. coli nonexpression strains. Saporin L3 is >10(3) times more efficient at RNA deadenylation on short GAGA stem-loops than saporin S6, the saporin isoform currently used in immunotoxin clinical trials. We engineered a construct for the His-tagged saporin L3 to test for expression in Pichia pastoris when it is linked to the protein export system for the yeast ?-mating factor. DNA encoding saporin L3 was cloned into a pPICZ?B expression vector and expressed in P. pastoris under the alcohol dehydrogenase AOX1 promoter. A fusion protein of saporin L3 containing the pre-pro-sequence of the ?-mating factor, the c-myc epitope, and the His tag was excreted from the P. pastoris cells and isolated from the culture medium. Autoprocessing of the ?-mating factor yielded truncated saporin L3 (amino acids 22-280), the c-myc epitope, and the His tag expressed optimally as a 32 kDa construct following methanol induction. Saporin L3 was also expressed with specific alanines and/or serines mutated to cysteine. Native and Cys mutant saporins are kinetically similar. The recombinant expression of saporin L3 and its mutants permits the production and investigation of this high-activity ribosome-inactivating protein.
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Project description:Polynucleotide kinase/phosphatase (PNKP) is a critical mammalian DNA repair enzyme that generates 5'-phosphate and 3'-hydroxyl groups at damaged DNA termini that are required for subsequent processing by DNA ligases and polymerases. The PNKP phosphatase domain recognizes 3'-phosphate termini within DNA nicks, gaps, or at double- or single-strand breaks. Here we present a mechanistic rationale for the recognition of damaged DNA termini by the PNKP phosphatase domain. The crystal structures of PNKP bound to single-stranded DNA substrates reveals a narrow active site cleft that accommodates a single-stranded substrate in a sequence-independent manner. Biochemical studies suggest that the terminal base pairs of double-stranded substrates near the 3'-phosphate are destabilized by PNKP to allow substrate access to the active site. A positively charged surface distinct from the active site specifically facilitates interactions with double-stranded substrates, providing a complex DNA binding surface that enables the recognition of diverse substrates.
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