Fig. 6: The bolt-action mechanism of proofreading.
a Primer-template scaffolds with a nick or a 10-nt gap between the primer and downstream DNA duplex barrier used in (b, c). b Primer extension assays performed with WT Polγ in the presence of all dNTPs on scaffolds shown in (a). c Exonuclease assay performed with WT Polγ on scaffolds having a nick or 10-nt gap and a 3′ end mismatched (MM) base. d Exonuclease assays performed with WT Polγ and Polγ_A. e–g Structural elements involved in proofreading in Polγ are conserved in the Pol A family of DNAPs. Close-up views of the major structural elements in Klenow (PDB 1KLN) (e), T7 DNAP (PDB 2AJQ) (f), and TAQ DNAP (PDB 4KTQ) (g) are shown. Structures are aligned with respect to their conserved palm subdomains. Primer-template DNA in the exo site (f, g) was modeled from the structure of the Klenow fragment (1KLN). Note that the existing T7 and TAQ structures do not reflect the proofreading step at which engagement of the G-loop is expected. Gels in b–d are representative results from triplicate experiments.
