ATP‐ and NAD⁺‐dependent DNA ligases share an essential function in the halophilic archaeon Haloferax volcanii

Abstract

SummaryDNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD + + + + -dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD + + + + -dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.

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

“…Phylogenetic analysis of the sequences presented here is consistent with previous studies of DNA ligases (Nakatani et al 2000). However, the addition of amino acid sequences for FaLig and related enzymes from other Thermoplasmatales identified that these are more similar to DNA ligases that have been characterized from Crenarchaeota (Jeon and Ishikawa, 2003;Kletzin 1992;Lai et al 2002) compared to those from Euryarchaeota (Gunther et al 2002;Keppetipola and Shuman 2005;Nakatani et al 2000;Rolland et al 2004;Sriskanda et al 2000;Zhao et al 2006). This phylogenetic description suggests that lateral gene transfer has occurred in the evolution of the DNA ligases of the Euryarchaeota.…”

“…The majority of archaeal DNA ligases that have been experimentally analysed use ATP as the co-factor for ligation, although those from some Euryarchaeota can also use other nucleotides (Jeon and Ishikawa 2003;Nakatani et al 2000;Rolland et al 2004;Zhao et al 2006). To assess the situation with FaLig, we analysed nick-joining in the presence of a variety of cofactors (Fig.…”

“…The same range of substrates was ligated by features Tomkinson et al 2006;Wilkinson et al 2001). Recently, however, characterization of DNA ligases from some archaea have identified unexpected features, particularly in relation to the role of the co-factor in enzyme function (Jeon and Ishikawa 2003;Nakatani et al 2000;Rolland et al 2004;Zhao et al 2006). We have cloned and characterized the single DNA ligase encoded by the genome of F. acidarmanus Fer1, an archaeon that is viable at extremely acidic conditions.…”

“…The ability for DNA ligases to use both ATP or NAD + as a cofactor appears to be specific to DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the Euryarchaeota (Rolland et al 2004). The relationship between ATP-and NAD + -dependent DNA ligases has taken a further twist upon analysis of Haloferax volcanii, since this organism encodes both types of protein that share the essential DNA-joining functions (Zhao et al 2006). …”

The Hyperthermophilic Euryarchaeon Archaeoglobus fulgidus Repairs Uracil by Single-Nucleotide Replacement

“…Bacterial DNA ligases use NAD and the mammalian enzymes use ATP for adenylation (32). Although there are exceptions (62,71), the archaeal ligases are considered ATP dependent and exhibit sequence homology with eukaryotic enzymes (12), and some of them have also been demonstrated to utilize ADP with similar efficiency (25). This is consistent with our results, which showed that addition of NAD increased the level of repaired DNA to a level somewhat greater than the control level, while addition of ATP and ADP resulted in a Ͼ1-order-of-magnitude increase in the level of the repair product following incubation for 60 min (Fig.…”