Polynucleotide 3′-terminal Phosphate Modifications by RNA and DNA Ligases [RNA]

October 16th, 2014 by Zhelkovsky, A. M., McReynolds, L. A.

ABSTRACT RNA and DNA ligases catalyze formation of phosphodiester bond between 5′-phosphate and 3′-hydroxyl ends of nucleic acids. In this work we describe the ability of the thermophilic RNA ligase, MthRnl from Methanobacterium thermoautotrophicum, to recognize and modify the 3′-terminal phosphate of RNA and single-stranded DNA. This ligase can use an RNA 3′p substrate to generated an RNA 2′,3′-cyclic phosphate or convert DNA3′p to ssDNA3′pp5′A. An RNA ligase from Thermus scotoductus bacteriophage TS2126, and a predicted T4 Rnl1-like protein from Thermovibrio ammonificans, TVa, were also able to adenylate DNA 3′p. These modifications of RNA and DNA 3′-phosphates are similar to the activities of RtcA, an RNA 3′-phosphate cyclase. The initial step involves adenylation of the enzyme by ATP, which is then transferred to either RNA 3′p or DNA 3′p to generate the adenylated intermediate. For RNA 3′pp5′A the third step involves attack of the adjacent 2′ hydroxyl to generate the RNA 2′,3′-cyclic phosphate. These steps are analogous to those in classical 5′ phosphate ligation. None of the RNA ligases tested were able to modify a 3′p in nicked dsDNA, however, T4 DNA ligase and RtcA can use 3′-phosphorylated nicks in dsDNA to produce 3′-adenylated product. These 3′-terminal phosphate adenylated intermediates are substrates for deadenylation by yeast 5′Deadenylase. Our findings that classic 5′pRNA ligases can duplicate the adenylation and phosphate cyclization activity of RtcA suggests that they have an essential role in metabolism of nucleic acids with 3′-terminal phosphates