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Nucleic Acids Res. 2014 Feb;42(3):1831-44. doi: 10.1093/nar/gkt1032. Epub 2013 Nov 6.

Efficient DNA ligation in DNA-RNA hybrid helices by Chlorella virus DNA ligase.

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  • 1DNA 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.

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.

PMID:
24203707
[PubMed - indexed for MEDLINE]
PMCID:
PMC3919565
Free PMC Article
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