The proteome of the amoebo-flagellate protozoan Naegleria gruberi is rich in candidate RNA repair enzymes， including 15 putative RNA ligases， one of which， NgrRnl， is a eukaryal homolog of Deinococcus radiodurans RNA ligase， DraRnl. Here we report that purified recombinant NgrRnl seals nicked 3'-OH/5'-PO4 duplexes in which the 3'-OH strand is RNA. It does so via the "classic" ligase pathway， entailing reaction with ATP to form a covalent NgrRnl-AMP intermediate， transfer of AMP to the nick 5'-PO4， and attack of the RNA 3'-OH on the adenylylated nick to form a 3'-5' phosphodiester. Unlike members of the four known families of ATP-dependent RNA ligases， NgrRnl lacks a carboxy-terminal appendage to its nucleotidyltransferase domain. Instead， it contains a defining amino-terminal domain that we show is important for 3'-OH/5'-PO4 nick-sealing and ligase adenylylation， but dispensable for phosphodiester synthesis at a preadenylylated nick. We propose that NgrRnl， DraRnl， and their homologs from diverse bacteria， viruses， and unicellular eukarya comprise a new "Rnl5 family" of nick-sealing ligases with a signature domain organization.