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The addition of Fen1 stimulated Pol-β activity for strand-displacement synthesis (Fig. 5C, lane 4), which was also ligated with DNA ligase I to generate 63-mer repaired product (Fig. 5C, lane 6).
The results showed efficient 1-nt incorporation (24-mer product) by Pol-β which was ligated with DNA ligase I to generate 63-mer repaired product (Fig. 5C, lane 5).
The quantification of intermediates and fully repaired product for each of the AlkB reactions above provided some additional insights.
Later studies by Yang et al. showed that prereduced SPL could catalyze at least 10 turnovers of SP to repaired product, supporting a catalytic role for SAM.
These additional reaction pathways could explain the improved yield of fully repaired product, dG, when NaBH4-treated M1dG was incubated with AlkB.
Subsequently, M1dG (7) reacted with AlkB, as shown before, to generate epoxide 8, triol 10, and, upon spontaneous loss of 2-hydroxy-MDA, the fully repaired product, dG.
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Thus, the 34-mer ligated products are fully repaired products of SP BER (Fig. 9B).
An adenine base was then added to the purified end repaired products using Klenow exo (3′ to 5′ exo minus) enzyme.
A time course showing the amount of repaired products of a G-I heteroduplex when incubated with HeLa extracts is shown in Fig. 2c, d.
Using the parallel tagged sequencing method described by Meyer et al. [ 57], palindromic 20-bp DNAs, whose sequences differ from species to species, were ligated to both 5' and 3' ends of the repaired products.
At first, we take a look at the economic barriers that dissuade consumers from repairing products.
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Justyna Jupowicz-Kozak
CEO of Professional Science Editing for Scientists @ prosciediting.com