Substrate recognition and fidelity of strand joining by an archaeal DNA ligase

Abstract

We have previously identi®ed a DNA ligase (Lig Tk ) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. The enzyme is the only characterized ATP-dependent DNA ligase from a hyperthermophile, and allows the analysis of enzymatic DNA ligation reactions at temperatures above the melting point of the substrates. Here . Using various 3¢ hydroxyl group donors (L-DNA) and 5¢ phosphate group donors (R-DNA), we could detect ligation products as short as 16 nucleotides, the products of 7 + 9 nucleotide or 8 + 8 nucleotide combinations at 40°C. An elevation in temperature led to a decrease in reaction eciency when short oligonucleotides were used, suggesting that the formation of a nicked, double-stranded DNA substrate preceded enzymesubstrate recognition. Lig Tk was not inhibited by the addition of excess duplex DNA, implying that the enzyme did not bind strongly to the double-stranded ligation product after nick-sealing. In terms of reaction ®delity, Lig Tk was found to ligate various substrates with mismatched basepairing at the 5¢ end of the nick, but did not show activity towards the 3¢ mismatched substrates. Lig Tk could not seal substrates with a 1-nucleotide or 2-nucleotide gap. Small amounts of ligation products were detected with DNA substrates containing a single nucleotide insertion, relatively more with the 5¢ insertions. The results revealed the importance of proper base-pairing at the 3¢ hydroxyl side of the nick for the ligation reaction by Lig Tk .Keywords: archaea; DNA ligase; hyperthermophile; Thermococcus.DNA ligases (EC 6.5.1.1 and EC 6.5.1.2) are universally found in bacteria, eukaryotes and archaea. In addition, they are also found in viruses and bacteriophages [1±5]. DNA ligases catalyse the phosphodiester bond formation between adjacent 3¢ hydroxyl and 5¢ phosphate groups at a singlestrand break in double-stranded DNA [5,6]. They are essential enzymes for maintaining the integrity of the genome during DNA replication [7], DNA excision repair [8] and DNA recombination [9]. DNA strand breaks are commonly generated as reaction intermediates in these events, and the sealing of these breaks depends solely on the proper function of DNA ligase [2]. Therefore DNA ligases are indispensable enzymes in all organisms.DNA ligases fall into two groups, ATP-dependent DNA ligases and NAD + -dependent DNA ligases, on the basis of the required cofactor for ligase±adenylate formation [2,5,6].ATP-dependent enzymes have been found in viruses, bacteriophages, eukaryotes, archaea and, recently, in bacteria, whereas NAD + -dependent enzymes have been found exclusively in bacteria [2,3,5]. There is high similarity among the ligases within the ATP-dependent groups [10] or NAD + -dependent groups [11,12]. However, enzymes between the two groups show no similarity, with the exception of the AMP-binding site [10]. It is now accepted that both ATP-dependent and NAD + -dependent DNA ligases catalyse their reactions through a common mechanism [13]. The ligation reaction proceeds through three steps. In the ®rst ...

Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions

“…Different DNA ligases display distinct patterns of fidelity, with respect to how well the ligase discriminates and the hierarchy of which mispairs are or are not accepted by the ligase (Tomkinson et al 1992;Husain et al 1995;Shuman 1995;Luo et al 1996;Sriskanda and Shuman 1998;Bhagwat et al 1999;Tong et al 2000;Nakatani et al 2002;Lamarche et al 2005;Wang et al 2007a). The consistent theme is that DNA ligase fidelity is greatest on the 3 ′ -OH side of the nick.…”

Profiling the selectivity of DNA ligases in an array format with mass spectrometry

“…We assayed each of the four substrates having an insertion of the four nucleotides at the nick site and found that none of these were adenylated by the ligase enzyme. Ligation of substrates with nucleotide insertions on 3′-side of the nick has been investigated for T. kodakaraensis, A. archaeal and T. thermophilus DNA ligases, and also showed negligible ligation activities (4,43,44). It has been also reported that DNA ends with nucleotide protrusion can be ligated to blunt ends by T4 DNA ligase under certain conditions, but as expected with low efficiency (45,46).…”

Effects of 3′-OH and 5′-PO 4 Base Mispairs and Damaged Base Lesions on the Fidelity of Nick Sealing by Deinococcus radiodurans RNA Ligase

“…1B and 2B). In contrast, DNA ligases are generally biased in their fidelity of mispair sealing, being highly sensitive to 3=-OH mispairs and comparatively tolerant of 5=-PO 4 mispairs (44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54).…”