Efficient DNA ligation in DNA–RNA hybrid helices by Chlorella virus DNA ligase
| Autor: | Yinhua Zhang, Eric J. Cantor, Alexander M. Zhelkovsky, Thomas C. Evans, Gregory J. S. Lohman |
|---|---|
| Rok vydání: | 2013 |
| Předmět: |
chemistry.chemical_classification
DNA ligase DNA Ligases Okazaki fragments Nucleic Acid Enzymes Circular bacterial chromosome DNA Biology Molecular biology Sequencing by ligation Kinetics Viral Proteins chemistry.chemical_compound chemistry Genetics RNA Ligation Ligase chain reaction DNA polymerase mu |
| Zdroj: | Nucleic Acids Research |
| ISSN: | 1362-4962 0305-1048 |
| DOI: | 10.1093/nar/gkt1032 |
| Popis: | 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 NaCl100 mM. Abortive adenylylation was suppressed at low ATP concentrations (100 µM) and pH8, 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. |
| Databáze: | OpenAIRE |
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