Journal: Molecular and Cellular Biology
Article Title: Polymerase Mu Is a DNA-Directed DNA/RNA Polymerase
Figure Lengend Snippet: pol μ ribonucleotide incorporation with mixed nucleotide pools. (A) Alkali cleavage assay for incorporation of ribonucleotides. A single-nucleotide-gapped DNA substrate with template C was 5′ 32 P labeled as indicated (star). Alkali treatment of products containing incorporated ribonucleotides results in product cleavage, as well as transfer of the 32 P from the 20-nt downstream strand to the primer strand, generating a novel 26-nt species (species II). (B) DNA substrate (5 nM) as in panel A was present in all reaction mixtures. Where indicated, 0.05 U of T4 ligase (+) and 0.5 nM pol μ (+) or 5 pM pol β (β) were added. Following a 1-min polymerization-ligation reaction, samples were treated with alkali as noted (+). A 25 μM concentration of each dNTP (d) or rNTP (r) was present as noted. Reaction mixtures 8 to 10 contained a mixture of both dNTPs and rNTPs (d + r), with 25 μM each dNTP and 25 μM (lane 8), 125 μM (lane 9), or 500 μM (lane 10) each rNTP. I, species I; II, species II.
Article Snippet: One unit of T4 ligase (Roche) was equivalent to ∼350 μg of X4-LIV.
Techniques: Cleavage Assay, Labeling, Ligation, Concentration Assay