Structure-Specific Endonucleolytic Cleavage of Nucleic Acids by Eubacterial DNA Polymerases

TLDR

The 5′ nuclease of DNA polymerase can specifically cleave any linear single‑stranded nucleic acid when a complementary oligonucleotide converts the target site into a substrate. The study shows that eubacterial DNA polymerase 5′ exonucleases act as structure‑specific endonucleases, cleaving ssDNA or RNA at duplex bifurcations independently of synthesis, with primers accelerating the reaction and allowing cleavage of 5′ arms up to 200 nt.

Abstract

Previously known 5′ exonucleases of several eubacterial DNA polymerases have now been shown to be structure-specific endonucleases that cleave single-stranded DNA or RNA at the bifurcated end of a base-paired duplex. Cleavage was not coupled to synthesis, although primers accelerated the rate of cleavage considerably. The enzyme appeared to gain access to the cleavage site by moving from the free end of a 5′ extension to the bifurcation of the duplex, where cleavage took place. Single-stranded 5′ arms up to 200 nucleotides long were cleaved from such a duplex. Essentially any linear single-stranded nucleic acid can be targeted for specific cleavage by the 5′ nuclease of DNA polymerase through hybridization with an oligonucleotide that converts the desired cleavage site into a substrate.