A DNA Ligase from a Hyperthermophilic Archaeon with Unique Cofactor Specificity

Abstract

A gene encoding DNA ligase (lig Tk ) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, has been cloned and sequenced, and its protein product has been characterized. ؉ . This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.DNA ligases (EC 6.5.1.1 and EC 6.5.1.2) catalyze the phosphodiester bond formation between adjacent 3Ј-hydroxyl and 5Ј-phosphoryl groups at a single-strand break in doublestranded DNA (22). They are essential enzymes for maintaining the integrity of the genome during DNA replication (24), DNA excision repair (48), and DNA recombination (16). DNA strand breaks are commonly generated as reaction intermediates in these events, and the sealing of these breaks solely depends on the proper function of DNA ligase. Therefore, DNA ligases are indispensable enzymes in all organisms.DNA ligases fall into two groups on the basis of the required cofactor for activity: the group requiring ATP (8) and the group requiring NAD ϩ (43). There is high similarity among the ligases within the ATP-dependent group (19) or NAD ϩ -dependent group (42). ATP-dependent DNA ligase I from humans and Saccharomyces cerevisiae are 42% identical, and NAD ϩ -dependent enzymes from Escherichia coli and Thermus thermophilus are 46% identical. However, enzymes between the two groups show no similarity, with the exception of the KXDG motif, which includes the active-site lysine (19). Furthermore, biochemical investigations have indicated that there is strict specificity towards the respective cofactors. This suggests that the two groups have evolved through completely different pathways.It is now accepted that both ATP-dependent and NAD ϩ -dependent DNA ligases catalyze their reactions through a common mechanism (7). The ligation reaction proceeds through three steps: (i) activation of the enzyme through the covalent addition of AMP to the conserved active-site lysine of the protein, accompanied by the release of PP i or nicotinamide mononucleotide from the cofactor (ATP or NAD ϩ ), (ii) transfer of AMP from the protein to the 5Ј-phosphoryl group of the nick on the DNA, and (iii) phosphodiester bond formation with concomitant release of free AMP from the adenylated DNA intermediate.Biochemical and genetic studies have been performed for DNA ligases from various organisms. It has been shown that eukaryotes (17, 33, 46), viruses (29, 30), and bacteriophages (7,20) harbor ATP-dependent enzymes. Although only a single type of DNA ligase has been reported for viruses and bacteriophages, eukaryotic organisms have multiple enzymes. Five distinct DNA ligases have been reported from mammalian cells (17,33,45,46). ATP-dependent enzymes show a wide range in molecular mass, from 41 kDa (bacteriophage T7) (7) to 102 kDa (human DNA ligase I) (2). This considerable difference in size is mainly due to the diversity of the N-terminal region of each DNA ligase. DNA ligase I from humans includes a regulatory domain of 216 amino acid residues in its N-terminal region, which is dispensable for DNA ligas...

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

“…To allow comparison with ATP-dependent DNA ligases from other organisms, sequences from human DNA ligase I and bacteriophages T4 and RB43 were also included. In agreement with an earlier, more-detailed phylogenetic description (Nakatani et al 2000), our analysis confirms that all archaeal ATP-dependent DNA ligases have a closer evolutionary relationship to each other than to other Extremophiles (2007) 11:315-327 319 ATP-dependent DNA ligases. In addition, our analysis shows that Thermoplasmatales are grouped in a clade that is separated from the other Euryarchaeota but branches with the Crenarchaeota (Fig.…”

“…1c). ATP-dependent DNA ligases and the classical bacterial NAD + -dependent enzymes are distant in evolutionary terms (Nakatani et al 2000). Thus, the amino acid sequences of the NAD + -dependent DNA ligases from E. coli and Streptomyces coelicolor (Wilkinson et al 2003) were used as an out-group to improve confidence in the phylogenetic description.…”

“…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 first archaeal DNA ligase was identified in 1992 from a hyperthermophilic Crenarchaeon, Delsulfurolobus ambivalens, with the amino acid sequence of this protein being most similar to those of Eukaryotic viral and cellular ATP-dependent ligases (Kletzin 1992;Nakatani et al 2000). This is not particularly surprising since Archaea have been shown to be similar to Eukarya in many aspects of DNA metabolism, despite their morphological and structural resemblance to Bacteria (Kelman and White 2005;White 2003).…”

“…The observation of some unexpected characteristics provides increasing interest in this class of proteins. For example, ATP-dependent DNA ligases from Thermococcus kodakaraensis (Nakatani et al 2000) and T. fumicolans (Rolland et al 2004) are also able to use NAD + as the cofactor for ligation. 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).…”