Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

Article Snippet: [γ–32 P]ATP (3000 Ci/mmol), [α–32 P]dATP (3000 Ci/mmol) and [α–32 P]dTTP (3000 Ci/mmol) were obtained from Amersham International Plc.

Techniques: Inhibition, Incubation, Concentration Assay

Journal: The EMBO Journal

Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

doi: 10.1093/emboj/19.7.1731

Figure Lengend Snippet: Fig. 2. Expression of human Pol μ in Escherichia coli . ( A ) Coomassie Blue staining after SDS–PAGE separation of control non-induced (NI) and IPTG-induced (I) extracts of E.coli BL21(DE3) cells transformed with the recombinant plasmid pRSET-hPolμ, and further purification steps of the latter extracts. The mobility of the induced protein Pol μ was compatible with its deduced molecular mass (55 kDa/494 amino acids). After PEI precipitation of the DNA, Pol μ was precipitated with 50% ammonium sulfate (AS), and purified further by phosphocellulose (PC) and heparin–Sepharose (HS) chromatography, as described in Materials and methods. The electrophoretic migration of a collection of molecular mass markers (MW) is shown at the left. ( B ) Relative activation by Mg 2+ versus Mn 2+ of TdT and Klenow enzymes during DNA polymerization ([α– 32 P]dATP labelling) on activated DNA. TdT (5 U) and Klenow (1 U) were assayed for 30 min at 37°C, in the presence of either 10 mM MgCl 2 or 1 mM MnCl 2 as a source of activating metal ions. DNA polymerase activity, expressed as dAMP incorporation, was quantitated as described in Materials and methods. ( C ) DNA polymerization activity associated with Pol μ expression. The 50% AS fraction corresponding to either non-induced (N.I.) or induced extracts was assayed and quantitated as described in ( B ).

Article Snippet: [γ–32 P]ATP (3000 Ci/mmol), [α–32 P]dATP (3000 Ci/mmol) and [α–32 P]dTTP (3000 Ci/mmol) were obtained from Amersham International Plc.

Techniques: Expressing, Staining, SDS Page, Transformation Assay, Recombinant, Plasmid Preparation, Purification, Chromatography, Migration, Activation Assay, Activity Assay