title: Engineered DNA ligases with improved activities in vitro.
abstract: The DNA ligase from bacteriophage T4 is one of the most widely used enzymes in molecular biology. It has evolved to seal single-stranded nicks in double-stranded DNA, but not to join double-stranded fragments with cohesive or blunt ends. Its poor activity in vitro, particularly with blunt-ended substrates, can lead to failed or sub-optimal experimental outcomes. We have fused T4 DNA ligase to seven different DNA-binding proteins, including eukaryotic transcription factors, bacterial DNA repair proteins and archaeal DNA-binding domains. Representatives from each of these classes improved the activity of T4 DNA ligase, by up to 7-fold, in agarose gel-based screens for cohesive- and blunt-ended fragment joining. Overall, the most active variants were p50-ligase (i.e. NF-κB p50 fused to T4 DNA ligase) and ligase-cTF (T4 DNA ligase fused to an artificial, chimeric transcription factor). Ligase-cTF out-performed T4 DNA ligase by ∼160% in blunt end 'vector + insert' cloning assays, and p50-ligase showed an improvement of a similar magnitude when it was used to construct a library for Illumina sequencing. The activity of the Escherichia coli DNA ligase was also enhanced by fusion to p50. Together, these results suggest that our protein design strategy is a generalizable one for engineering improved DNA ligases.
authors.affiliation: Institute of Natural Sciences, Massey University, Auckland 0632, New Zealand.
authors.forename: Robert H
authors.initials: RH
authors.lastname: Wilson
authors.forename: Susan K
authors.initials: SK
authors.lastname: Morton
authors.forename: Heather
authors.initials: H
authors.lastname: Deiderick
authors.forename: Monica L
authors.initials: ML
authors.lastname: Gerth
authors.forename: Hayden A
authors.initials: HA
authors.lastname: Paul
authors.forename: Ilana
authors.initials: I
authors.lastname: Gerber
authors.forename: Ankita
authors.initials: A
authors.lastname: Patel
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Ellington
authors.forename: Scott P
authors.initials: SP
authors.lastname: Hunicke-Smith
authors.forename: Wayne M
authors.initials: WM
authors.lastname: Patrick
mesh_terms: D017122:Bacteriophage T4 / Q000201:enzymology*; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D011088:DNA Ligases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D011088:DNA Ligases
keywords: DNA ligase; DNA-binding protein; enzyme engineering; fusion protein