Figure 3.

Reconstitution of flap removal in a coupled replication-ligation assay. (A) Schematic of assay showing the flap-template. Per reaction, 10 fmol of template was incubated with mtSSB, and where indicated, POLγ, T4 DNA ligase and MGME1 (300 fmol). Products were labeled with [α-³²P] dCTP during the reaction and run on agarose gels containing EtBr to separate closed circular (ligated) products from nicked products. (B) MGME1 is required for ligation in the presence of a 5′-primer flap. Double stranded nicked products (nicked arrowhead) are formed after DNA synthesis. Supercoiled (ligated arrowhead) DNA is only formed in the presence of MGME1 and T4 DNA ligase. Templates are shown above the gel: 5′-flap primed template (lanes 1–5); control 5′-phosphorylated (P) fully annealed primed template (lanes 6–10). (C) As in (B), but reconstitution of mtDNA replication using only human mitochondrial enzymes, specifically DNA ligase 3 instead of T4 DNA ligase. (D) The 3′-5′ exonuclease activity of POLγ is required for ligation. Essentially same assay as in (B), but using either WT POLγ or EXO- POLγ. The flap template is shown above.