Translesion DNA Synthesis Mechanisms

All materials, including reagents and solvents, were of commercial grade. [γ-³²P]-ATP was from Perkin Elmer Health Sciences Inc. (Shelton, CT). The enzymes and solutions of native dNTPs were purchased from New England Biolabs (Beverly, MA). HEK 293T cells with knockout of a single TLS polymerase hPol η, hPol κ, hPol ι, and hPol ζ, and simultaneous knockout of hPol κ/hPol ι were a gift from Professor Yinsheng Wang (University of California, Riverside, CA), which were produced by using the CRISPR-Cas9 genome editing method.^{41 (link),42 (link)} Synthetic siRNA duplexes against hPol λ (SI00101108) and negative control siRNA (1027280) were purchased from Qiagen (Valencia, CA). Oligonucleotides containing 8-OxodGuo were prepared using commercially available phosphoramidites (Glen Research), deprotected using manufacturer recommended protocols, and purified by denaturing PAGE. Purified oligonucleotides were characterized by MALDI-TOF MS (Figure S1). PAGE analysis indicated that they are >96% pure. Unmodified oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA).