Immobilized DNA hairpins for assay of sequential breaking and joining of DNA backbones.
Immobilized DNA hairpins are exploited in a novel approach to assay DNA ligases and nucleases. A fundamental characteristic of the assay is that a fluorophore at the remote terminus of the hairpin reports on the integrity of the DNA backbone. The functionality of the protocol is confirmed using ATP- and NAD+-dependent DNA ligases and the nicking enzyme N.BbvCIA. The assay format is amenable to high-throughput analysis and quantitation of enzyme activity, and it is shown to be in excellent agreement with the more laborious electrophoretic approaches that are widely used for such analyses. Significantly, the assay is used to demonstrate sequential breaking and rejoining of a specific nucleic acid. Thus, a simple platform for biochemically innovative studies of pathways in cellular nucleic acid metabolism is demonstrated.
Scott
Benjamin O S
BO
School of Chemical Sciences and Pharmacy, University of East Anglia, Norwich NR4 7TJ, UK.
Lavesa-Curto
Manuel
M
Bullard
Desmond R
DR
Butt
Julea N
JN
Bowater
Richard P
RP
D001483:Base Sequence; D001710:Biotin / Q000737:chemistry; D004247:DNA / Q000737:chemistry*; D011088:DNA Ligases / Q000032:analysis* / Q000378:metabolism; D004260:DNA Repair / Q000502:physiology*; D003850:Deoxyribonuclease I / Q000032:analysis*; D029968:Escherichia coli Proteins / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D019809:Streptavidin / Q000737:chemistry
D029968:Escherichia coli Proteins; D001710:Biotin; D004247:DNA; D019809:Streptavidin; D003850:Deoxyribonuclease I; D011088:DNA Ligases; C497118:LigA protein, E coli; D019793:Fluorescein