Specificity and fidelity of strand joining by Chlorella virus DNA ligase

Abstract

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.

Characterization of an ATP-dependent DNA ligase from the acidophilic archaeon “Ferroplasma acidarmanus” Fer1

“…After analysis on a denaturing polyacrylamide gel, the extent of nickjoining was quantitated Extremophiles (2007) 11:315-327 323 the 3¢-hydroxyl and deoxyribonucleotide containing the 5¢-phosphate. These effects on biochemical activity have been observed for other nucleic acid ligases, including human DNA ligase I (Pascal et al 2004), the bacterial cellular enzyme DraRnl from Deinococcus radiodurans (Martins and Shuman 2004) and viral enzymes Chlorella PBCV-1 DNA ligase (Sriskanda and Shuman 1998), Vaccinia DNA ligase (Sekiguchi and Shuman 1997) and T4 RNA ligase 2 Nandakumar and Shuman 2005). In most facets that we examined, FaLig was observed to be a rather typical ATP-dependent DNA ligase.…”

“…Recent studies identify that DNA ligases may join breaks in a variety of double-stranded nucleic acids (Martins and Shuman 2004;Pascal et al 2004;Sekiguchi and Shuman 1997;Sriskanda and Shuman 1998;Tomkinson et al 2006), with the range of active substrates varying compared to enzymes characterized as ''RNA'' ligases (Bullard and Bowater 2006). To assess how FaLig fitted into these categories, we analysed its nick-joining activity on a range of substrates.…”

Identification of a novel motif in DNA ligases exemplified by DNA ligase IV

“…6b) and actively maintains the B-form that is essential for efficient ligation. Consistent with this model, it has been shown that the presence of an A-form structure downstream of a nick severely affects ligation efficiency [17,23,24].…”

RNA Substrate Specificity and Structure-guided Mutational Analysis of Bacteriophage T4 RNA Ligase 2

“…Rnl2 displays "fidelity" in RNA nick joining comparable with that of DNA ligases in sealing nicked duplex DNAs. Like Rnl2, exemplary ATP-dependent DNA ligases are inhibited by displacement of the reactive termini by 1-or 2-nucleotide gaps and are typically more affected by perturbations at the 3Ј-OH terminus than at the 5Ј-PO 4 terminus (35)(36)(37)(38).…”