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. 2008 Aug 6;105(32):11122–11127. doi: 10.1073/pnas.0805399105

Fig. 5.

Fig. 5.

Lagging-strand DNA synthesis in vitro. (A) Minicircle template for rolling-circle DNA replication. Radioactive dNTPs can be used to preferentially label the synthesis of leading ([α-32P]dCTP) or lagging ([α-32P]dGTP) strands. The single G and C in the templates for lagging and leading strands are indicated with an asterisk. (B and C) Products formed by rolling-circle DNA replication in vitro were analyzed by electrophoresis on an alkaline agarose gel as described in Materials and Methods. The reaction mixtures contained POLγ (200 fmol), TWINKLE (100 fmol), mtSSB (5 pmol), and POLRMT (200 fmol) as indicated. (B) POLRMT supports lagging-strand DNA synthesis. Lane 1, DNA size markers; lanes 2 and 3, leading-strand DNA synthesis labeled with [α-32P]dCTP; lanes 4 and 5, lagging-strand DNA synthesis labeled with [α-32P]dGTP. (C) Southern blot analyses of replication products. In lanes 1 and 2, the oligonucleotide probe is complementary to the leading strand; in lanes 3 and 4, the oligonucleotide probe is complementary to the lagging strand.