title: Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.
abstract: Single-stranded DNA molecules (ssDNA) annealed to an RNA splint are notoriously poor substrates for DNA ligases. Herein we report the unexpectedly efficient ligation of RNA-splinted DNA by Chlorella virus DNA ligase (PBCV-1 DNA ligase). PBCV-1 DNA ligase ligated ssDNA splinted by RNA with kcat ≈ 8 x 10(-3) s(-1) and K(M) < 1 nM at 25 °C under conditions where T4 DNA ligase produced only 5'-adenylylated DNA with a 20-fold lower kcat and a K(M) ≈ 300 nM. The rate of ligation increased with addition of Mn(2+), but was strongly inhibited by concentrations of NaCl >100 mM. Abortive adenylylation was suppressed at low ATP concentrations (<100 µM) and pH >8, leading to increased product yields. The ligation reaction was rapid for a broad range of substrate sequences, but was relatively slower for substrates with a 5'-phosphorylated dC or dG residue on the 3' side of the ligation junction. Nevertheless, PBCV-1 DNA ligase ligated all sequences tested with 10-fold less enzyme and 15-fold shorter incubation times than required when using T4 DNA ligase. Furthermore, this ligase was used in a ligation-based detection assay system to show increased sensitivity over T4 DNA ligase in the specific detection of a target mRNA.
authors.affiliation: DNA Enzymes Division, New England BioLabs, Inc., Ipswich, MA 01938-2723, USA, RNA Biology Division, New England BioLabs, Inc., Ipswich, MA 01938-2723, USA and Applications Development, New England BioLabs, Inc., Ipswich, MA 01938-2723, USA.
authors.forename: Gregory J S
authors.initials: GJ
authors.lastname: Lohman
authors.forename: Yinhua
authors.initials: Y
authors.lastname: Zhang
authors.forename: Alexander M
authors.initials: AM
authors.lastname: Zhelkovsky
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Cantor
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Evans
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D011088:DNA Ligases / Q000378:metabolism*; D007700:Kinetics; D012313:RNA / Q000737:chemistry / Q000378:metabolism*; D014764:Viral Proteins / Q000378:metabolism*
chemical_list: D014764:Viral Proteins; D012313:RNA; D004247:DNA; C110456:Chlorella virus DNA ligase; D011088:DNA Ligases