Characterization of a Thermophilic ATP-Dependent DNA Ligase from the Euryarchaeon Pyrococcus horikoshii

Abstract

Archaea encode a DNA ligase composed of a C-terminal catalytic domain typical of ATP-dependent ligases plus an N-terminal domain similar to that found in eukaryotic cellular and poxvirus DNA ligases. All archaeal DNA ligases characterized to date have ATP-dependent adenylyltransferase and nick-joining activities. However, recent reports of dual-specificity ATP/NAD ؉ ligases in two Thermococcus species and Pyrococcus abyssi and an ATP/ADP ligase in Aeropyrum pernix raise the prospect that certain archaeal enzymes might exemplify an undifferentiated ancestral stage in the evolution of ligase substrate specificity. Here we analyze the biochemical properties of Pyrococcus horikoshii DNA ligase. P. horikoshii ligase catalyzes autoadenylylation and nick sealing in the presence of a divalent cation and ATP; it is unable to utilize NAD ؉ or ADP to promote ligation in lieu of ATP. P. horikoshii ligase is thermophilic in vitro, with optimal adenylyltransferase activity at 90°C and nick-joining activity at 70 to 90°C. P. horikoshii ligase resembles the ligases of Methanobacterium thermautotrophicum and Sulfolobus shibatae in its strict specificity for ATP.DNA ligases are essential components of the DNA replication, repair, and recombination machinery in all domains of the phylogenetic tree: eucarya, archaea, and bacteria. Ligases seal 3Ј OH and 5Ј PO 4 ends via three nucleotidyl transfer reactions (10). First, a lysine nucleophile on the enzyme attacks the ␣-phosphorus of ATP or NAD ϩ , which results in the formation of a covalent ligase-adenylate intermediate and the release of pyrophosphate or nicotinamide mononucleotide. Second, attack by the 5Ј PO 4 on ligase-adenylate results in expulsion of the active-site lysine and formation of an activated DNA-adenylate intermediate. Third, ligase catalyzes the attack of a 3Ј OH on the DNA-adenylate, resulting in the release of AMP and formation of a phosphodiester.DNA ligases are grouped into two families, depending on their requirement for ATP or NAD ϩ in the ligase adenylylation reaction. Whereas NAD ϩ -dependent ligases are found only in bacteria and eukaryotic viruses, ATP-dependent DNA ligases are found in bacteria (and bacteriophages), eucarya (and eukaryotic viruses), and archaea (8,10,21,27). A core ligase module composed of nucleotidyltransferase and OB-fold domains is shared by all known DNA ligases (3,15,16,24). The adenylate-binding pocket is located within the nucleotidyltransferase domain and is composed of five conserved peptide motifs (19). The substrate specificity of NAD ϩ -dependent ligase is dictated by a unique domain that binds the nicotinamide mononucleotide component of the nucleotide substrate (3, 23); the specificity of ATP-dependent ligases is determined, at least in part, by a distinctive motif within the OB-fold domain that coordinates the beta and gamma phosphates of the nucleotide (5,19,22).The archaea are unicellular anucleate organisms with distinctive biosynthetic capacities and an ability to thrive under extreme environmental conditions. The dom...

Identification of a Novel Binding Motif in Pyrococcus furiosus DNA Ligase for the Functional Interaction with Proliferating Cell Nuclear Antigen

“…This result is the same as in the case of DNA ligase from Pyrococcus horikoshii in a recent report (21). We think that the ligation reaction may be derived from contaminant ATP (1.16%) in our ADP reagent (Oriental Yeast Co., Ltd., Osaka, Japan) according to the manufacturer's certificate, and therefore, we concluded that ADP is not an appropriate cofactor for PfuLig, as Keppetipola and Shuman (21) described for P. horikoshii DNA ligase.…”

“…In Archaea, the third domain of life, a single homolog of eukaryotic Lig I has been identified (17)(18)(19)(20)(21). Interestingly, although most of the archaeal replicative enzymes have a eukaryotic PIP box at their C termini, no clear PCNA-binding motif has been observed in the sequences of archaeal DNA ligases (5,22).…”

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 pH profile of FaLig is similar to that observed for a variety of DNA ligases, but it is now clear that the optimal pH for activity varies for enzymes from different organisms. In terms of archaea that grow at neutral pH, the optimal activity of their DNA ligases has been detected to be pH 7-8.5 (Jeon and Ishikawa 2003;Keppetipola and Shuman 2005;Rolland et al 2004;Sriskanda et al 2000). For DNA ligase from S. shibatae, which is able to grow at pH 2-4, optimal nickjoining was detected at pH 6-7 (Lai et al 2002).…”

DNA Ligases: Progress and Prospects

“…Biochemical characterization of several archaeal DNA ligases verified their strict dependence on ATP and failure to utilize NAD ϩ (summarized in Ref. 22). By contrast, other archaeal ligases were reported to have "dual specificity," variously using ATP and NAD ϩ or ATP and ADP (supplemental Fig.…”