Structure‐Based Mutational Study of an Archaeal DNA Ligase towards Improvement of Ligation Activity

Abstract

DNA ligases catalyze the joining of strand breaks in duplex DNA. The DNA ligase of Pyrococcus furiosus (PfuLig), which architecturally resembles the human DNA ligase I (hLigI), comprises an N-terminal DNA-binding domain, a middle adenylylation domain, and a C-terminal oligonucleotide-binding (OB)-fold domain. Here we addressed the C-terminal helix in the OB-fold domain of PfuLig by mutational analysis. The crystal structure of PfuLig revealed that this helix stabilizes a closed conformation of the enzyme by forming several ionic interactions with the adenylylation domain. The C-terminal helix is oriented differently in hLigI when DNA is bound; this suggested that disruption of its interaction with the adenylylation domain might facilitate the binding of DNA substrates. We indeed identified one of its residues, Asp540, as being critical for ligation efficiency. The D540R mutation improved the overall ligation activity relative to the wild-type enzyme, and at lower temperatures; this is relevant to applications such as ligation amplification reactions. Physical and biochemical analyses indicated that the improved ligation activity of the D540R variant arises from effects on the ligase adenylylation step and on substrate DNA binding in particular.

Structure of the Catalytic Region of DNA Ligase IV in Complex with an Artemis Fragment Sheds Light on Double-Strand Break Repair

“…Interestingly, the results of DNA ligation assays suggest that the residues 610–620 are important for the enzymatic activity of LigIV (Figure S1C). A recent report also suggests that the C-terminal helix of an archaeal DNA ligase is important for its activity (Tanabe et al., 2012). …”

Exonuclease processivity of archaeal replicative DNA polymerase in association with PCNA is expedited by mismatches in DNA

“…5A)24. When the ratio of the substrate DNA to PfuPCNA/PfuLig was relatively low, no facilitation of the nick-ligation was observed even with the wild type PfuPCNA, whereas the facilitation effect by PfuPCNA was detected in the presence of a large excess of the substrate.…”

“…The reactions were initiated by the addition of the enzymes, and were halted by the addition of a stop reagent (20 mM EDTA and 90% formamide). The samples were heated at 95 °C for 5 min and then electrophoresed through an 11% polyacrylamide gel containing 8 M urea in TBE (89 mM Tris/Tris-borate; 2 mM EDTA)2429. The quantities of the products in the ligation reaction were estimated from the fluorescent band intensities on the gel, obtained with a FluoroImager 595 (GE Healthcare).…”

Mutations of Asp540 and the domain‐connecting residues synergistically enhance Pyrococcus furiosus DNA ligase activity

“…We previously searched for an effective substitution in the C‐terminal extension helix region to improve the nick‐joining activity, and found that point mutations of Asp540, and especially the replacement with a basic residue (D540R), significantly enhanced the enzymatic activity. However, unexpectedly, D540R showed a much higher Δ H value than that of the wild type for substrate DNA binding, thus implying that the DNA‐binding process involves unfavorable bond disruption in D540R [13].…”