Journal:

Article Title: DNA polymerase ? (POLQ) can extend from mismatches and from bases opposite a (6-4) photoproduct

doi: 10.1016/j.dnarep.2007.08.005

Figure Lengend Snippet: A) Extension from primers having a mismatch. Exo-free Klenow fragment of E. coli pol I (9 x 10−4 Unit) was incubated in pol I buffer with the same substrates as in Fig. 1: A:T match (lanes 2–6), A:G mismatch (lanes 8–12), A:C mismatch (lanes 14–18) and T:T mismatch (lanes 20–24). Control reaction mixtures without DNA polymerase are in lanes 1, 7, 13, and 19.

Article Snippet: E. coli exo free pol I (Klenow fragment, Kf) was purchased from Promega Corporation, Madison, Wisconsin (catalog number M2181) and the supplied pol I buffer was used for the assays (50 mM Tris-HCl [pH 7.2], 10 mM MgSO 4 , 0.1 mM dithiothreitol).

Techniques: Incubation, Control

Journal: Nucleic Acids Research

Article Title: Novel alternative ribonucleotide excision repair pathways in human cells by DDX3X and specialized DNA polymerases

doi: 10.1093/nar/gkaa948

Figure Lengend Snippet: A role of DDX3X in ribonucleotide excision repair. ( A ) RER reactions in the presence of DDX3X (lanes 2–3), Pol β, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5, 7: re-digestion by RNaseH2 of the heat inactivated reactions. RER reactions in the presence of DDX3X, Pol δ, PCNA, dNTPs (lanes 8–9), Fen-1 and DNA ligase 1 (lanes 10–11). Lanes 9 and 11: re-digestion by RNaseH2. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( B ) RER reactions in the presence of DDX3X (lanes 2–3), Pol λ, dNTPs (lanes 4–5), Fen-1 and DNA ligase 1 (lanes 6–7). Lanes 3, 5 and 7: re-digestion by RNaseH2. Lane 1: Substrate * D 39 R 1 D 15 : D 55 alone. ( C ) Western blot for DDX3X and β-actin. L, molecular weight markers. NSC, non-silencing vector control cells; shDDX3X, stable DDX3X-silenced cells. ( D ) Quantification of genomic rNMPs by the riboassay. NSC untreated cells signal was set as 1 fluorescence arbitrary unit. Different treatments were: untreated genomic DNA (–), genomic DNA treated with 1 U of E. coli RNaseH2 or with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments. ( E ) Western blot for vinculin, DDX3X and RNaseH2A. L, molecular weight markers. siCNT, non-silencing smart-pool siRNAs; siDDX3X, DDX3X-silenced cells, siRH2A, RNaseH2A-silenced cells. ( F ) Quantification of genomic rNMPs by the riboassay. siCNT (control) cells signal was set as 1 fluorescence arbitrary unit. As above, different treatments were: untreated genomic DNA (–) and genomic DNA treated with 50 nM of human recombinant RNaseH2 (+). Data represent mean ± SEM of at least four independent experiments.

Article Snippet: Then, 1 μg of genomic DNA was digested with 1 U of E. coli RNaseH2 (BioLabs, Euroclone) or with 50 nM human recombinant RNaseH2 and the reaction was led in a dry heating block (Eppendorf ® Thermomixer ® ) at 37°C with constant shaking at 550 rpm for 2.5 h. Reaction was stopped on ice for 30 min and nick translation reaction started trough the addition of 1 U of E. coli DNA pol I (Biolabs, Euroclone) and 2 μl of Atto647N-dUTP pH 7.5 NT Labelling Kit (Jena Biosciences, Löbstedter, Germany) at 16°C for 1 h. Finally, the reaction was stopped by addition of standard gel loading buffer and loaded on a 1% agarose gel at 100 V for 2 h in TBE buffer.

Techniques: Western Blot, Molecular Weight, Plasmid Preparation, Fluorescence, Recombinant