T4 RNA ligase catalyzes the synthesis of dinucleoside polyphosphates

Abstract

T4 RNA ligase has been shown to synthesize nucleoside and dinucleoside 5 H -polyphosphates by displacement of the AMP from the E-AMP complex with polyphosphates and nucleoside diphosphates and triphosphates. Displacement of the AMP by tripolyphosphate (P 3 ) was concentration dependent, as measured by SDS/PAGE. When the enzyme was incubated in the presence of 0.02 mm [a-32 P] ATP, synthesis of labeled Ap 4 A was observed: ATP was acting as both donor (K m , mm) and acceptor (K m , mm) of AMP from the enzyme. Whereas, as previously known, ATP or dATP (but not other nucleotides) were able to form the E-AMP complex, the specificity of a compound to be acceptor of AMP from the E-AMP complex was very broad, and with K m values between 1 and 2 mm. In the presence of a low concentration (0.02 mm) of [a-32 P] ATP (enough to form the E-AMP complex, but only marginally enough to form Ap 4 A) and 4 mm of the indicated nucleotides or P 3 , the relative rate of synthesis of the following radioactive (di)nucleotides was observed: Ap 4 X (from XTP, 100); Ap 4 dG (from dGTP, 74); Ap 4 G (from GTP, 49); Ap 4 dC (from dCTP, 23); Ap 4 C (from CTP, 9); Ap 3 A (from ADP, 5); Ap 4 ddA, (from ddATP, 1); p 4 A (from P 3 , 200). The enzyme also synthesized efficiently Ap 3 A in the presence of 1 mm ATP and 2 mm ADP. The following T4 RNA ligase donors were inhibitors of the synthesis of Ap 4 G: pCp . pAp . pA2 H p.

Characterization of a Thermophilic ATP-Dependent DNA Ligase from the Euryarchaeon Pyrococcus horikoshii

“…For the reasons noted above in discussing why high concentrations of ATP were in- hibitory to steady-state ligation, the ADP inhibition cannot plausibly be attributed to sequestration of magnesium (present at 5 mM). The most likely explanation for the ADP inhibition of single-turnover ligation and ATP inhibition of steady-state ligation is that high concentrations of ATP and ADP can act as decoy acceptors for AMP transfer by ligase-adenylate, thereby diverting the ligase from nick joining and toward the synthesis of dinucleoside polyphosphates, as described by Sillero and colleagues (1,4,12). The Sillero lab has also shown that nucleoside triphosphates (NTPs) can act as decoy acceptors for AMP transfer.…”

“…Although neither NAD ϩ nor ADP could replace ATP as the nucleotide substrate for nick joining, ADP was uniquely inhibitory to single-turnover ligation by preformed ligase adenylate. Sillero et al (1,4,12) have shown that DNA and RNA ligases can donate their covalently bound adenylates to nonnucleic acid acceptors, particularly to NTPs or nucleoside diphosphates, thereby forming ApppN and AppppN dinucleotide products. The reaction mechanism is presumed to mimic the pyrophosphorolytic reversal of the step 1 ligase adenylylation reaction, which regenerates ATP (26).…”

Stereochemical Course ofEscherichia coli RNase H

“…This result, confirmed by MALDI-TOF MS, is in agreement with earlier reports that A P4 A can be nonspecifically synthesised by RNA ligase. [17] Not even the use of some agents that are known to increase efficiency of enzymatic ligation (bovine serum albumin, polyethylene glycol or Ficoll 70) [18] could increase the efficiency of this reaction. Further attempts to elucidate the stereochemistry of RNase H cleavage by the ligation approach were therefore abandoned.…”

“…His group initiated studies in chirotechnology, exemplified by the first synthesis of enantiomerically pure cyclophosphates of therapeutic relevance, such as cyclophosphamide, isophosphamide and trophosphamide. His contribution to the synthesis and stereochemistry of organophosphates includes the conformational analysis of dioxaphosphorinanes, stereospecific PN 3PX conversion demonstrated by the first stereospecific syntheses of R p -and S p -cAMPS and the design of a novel approach to stereospecific synthesis of diesters and amidoesters of phosphorothioic and isotopically labeled [ 16 O, 17 O, 18 O] phosphoric acids. His oxathiaphospholane approach to the synthesis of P-chiral organophosphates flourished in the first stereocontrolled synthesis of phosphorothioate oligonucleotides, a class of compounds first chemically synthesized by Stec et al and now broadly used in drug target validation and antisense therapy of viral and cancer diseases.…”

Recent progress in the study of the intracellular functions of diadenosine polyphosphates

“…T4 RNA ligase also transfers the adenylyl moiety of the E-AMP complex to a nucleoside triphosphate (NTP) giving rise to a dinucleoside tetraphosphate [Atencia et al, 1999]: 4.) E-AMP + NTP → Ap 4 N + E Because a dinucleoside polyphosphate resembles the 3′-OH end of an RNA, it could conceivably behave as an RNA acceptor analog in reaction 3.…”