RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends - PubMed (original) (raw)

Requirements for DNA 5′-adenylylation by RtcA. A, kinetic profile. The “complete” reaction mixture (130 μl) containing 50 m

m

Tris acetate (pH 6.0), 1 m

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DTT, 2 m

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MgCl2, 1 m

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ATP, 300 n

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5′-32P-labeled 12-mer DNA, and 80 n

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RtcA was incubated at 37 °C. Divalent cation and ATP were omitted from reaction mixtures, −Mg2+ and −ATP, respectively. Aliquots (10 μl, containing 3 pmol DNA) were withdrawn at the times specified and quenched immediately with formamide-EDTA. The time 0 sample was withdrawn prior to adding the RtcA. The extents of conversion of the pDNA strand to AppDNA product are plotted as a function of reaction time. Each datum is the average of three independent experiments ± S.E. (error bars). B, pH profile. Reaction mixtures (10 μl) containing either 50 m

m

Tris acetate (pH 4.5, 5.0, 5.5 6.0, or 6.5) or Tris-HCl (pH 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, or 9.5), 1 m

m

DTT, 2 m

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MgCl2, 1 m

m

ATP, 3 pmol 5′-32P-labeled 12-mer DNA, and 0.6 pmol RtcA were incubated at 37 °C for 30 min. The extents of conversion of the pDNA strand to AppDNA product are plotted as function of pH. Each datum is an average of three independent experiments ± S.E.