FIGURE 3.
Determination of bypass efficiencies and mutation frequencies of (S)- or (R)-N2-CEdG and N2-CMdG. A, restriction digestion of PCR products of the progeny genome arising from in vivo replication of N2-CEdG- and N2-CMdG-bearing genome in mammalian cells. The PCR fragments were digested with SacI, and the nascent terminal phosphate groups in the resulting DNA fragments were removed by shrimp alkaline phosphatase. The 5′-termini were labeled with [γ-32P]ATP, and the resulting DNA fragments were further digested with FspI. DNA fragments subsequently were resolved on a 30% native PAGE gel. B, nondenaturing PAGE for assessing the bypass efficiencies and mutation frequencies of N2-CEdG and N2-CMdG in mammalian cells that are proficient in TLS DNA polymerase (human 293T cells and wild-type MEF cells) or deficient in polymerase η (POLH−/−), polymerase ζ (Rev3−/−), polymerase κ (Polk−/−), and polymerase ι (Poli−/−).