title: Substrate recognition and fidelity of strand joining by an archaeal DNA ligase.
abstract: We have previously identified a DNA ligase (LigTk) 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 we have focused on the interactions of LigTk with various DNA substrates, and its specificities toward metal cations. LigTk could utilize Mg2+, Mn2+, Sr2+ and Ca2+ as a metal cation, but not Co2+, Zn2+, Ni2+, or Cu2+. The enzyme displayed typical Michaelis-Menten steady-state kinetics with an apparent Km of 1.4 microm for nicked DNA. The kcat value of the enzyme was 0.11*s-1. 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 degrees C. An elevation in temperature led to a decrease in reaction efficiency when short oligonucleotides were used, suggesting that the formation of a nicked, double-stranded DNA substrate preceded enzyme-substrate recognition. LigTk 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 fidelity, LigTk was found to ligate various substrates with mismatched base-pairing at the 5' end of the nick, but did not show activity towards the 3' mismatched substrates. LigTk 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 LigTk.
authors.affiliation: Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Japan.
authors.forename: Masaru
authors.initials: M
authors.lastname: Nakatani
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ezaki
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Atomi
authors.forename: Tadayuki
authors.initials: T
authors.lastname: Imanaka
mesh_terms: D020137:Base Pair Mismatch; D001483:Base Sequence; D002413:Cations, Divalent; D011088:DNA Ligases / Q000378:metabolism*; D017931:DNA Primers; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D019712:Thermococcus / Q000201:enzymology*
chemical_list: D002413:Cations, Divalent; D017931:DNA Primers; D011994:Recombinant Proteins; D011088:DNA Ligases