Figure 5.

Impact of the generation of sequence expansions during NHEJ repair reactions by Polµ. (A) The scheme corresponds to the end-joining substrates used, whose 3′-protrusions can be connected by three bases pairs but leaving a distortion (1 flipped-out base) close to the 3′-primer terminus. Such a connection leaves two different 1-nt gaps. Gap-filling of one of them (that flanked by a 5′-P) is evaluated as a function of each possible templating base (X). Thus, the 5′-labelled substrate (dark grey) will be tested as primer, whereas the cold substrate (light grey; in which the X in the scheme is changed to A, C, G or T) is providing the template for the connection. Polymerization reactions were performed in the presence of 200 nM Polµ, 2.5 mM MgCl₂ and 100 µM of a single dNTP (complementary to X in each case). After incubation for 1 h at 30°C, reactions were stopped and loaded on 20% PA-8 M urea gels. Labelled DNA fragments were detected by autoradiography. (B) Polymerization reactions performed as in (A), but using end-joining substrates used whose 3′-protrusions can be connected by three bases pairs with no distortion.