Table 3.
Effects of Y2387A, Y2391A, or Y2387A/Y2391A mutations in Polθ on mutation frequencies and nucleotides inserted opposite the ϵdA adduct carried on the leading-strand DNA template in HFs (GM637)
| siRNA | Vector expressing | No. of Kan+ blue colonies sequenced | Nucleotide inserted |
Mutation frequency | Error-prone pathway that remains active | |||
|---|---|---|---|---|---|---|---|---|
| A | G | C | T | |||||
| % | ||||||||
| Polθ | FLAG-WT Polθ | 146 (16)a | 3 | 2 | 11 | 130 | 11.0 | Rev1 Polb |
| Polθ | FLAG-siRc-WT Polθ | 138 (21) | 5 | 1 | 15 | 117 | 15.2 | Rev1 Pol, Polθ |
| Polθ | FLAG-siR-Y2387A Polθ | 144 (9) | 1 | 0 | 8 | 135 | 6.3 | Rev1 Polb |
| Polθ | FLAG-siR-Y2391A Polθ | 156 (57) | 20 | 10 | 27 | 99 | 36.5 | Rev1 Pol, Y2391A Polθ |
| Polθ | FLAG-siR-Y2387A/Y2391A Polθ | 136 (9) | 1 | 0 | 8 | 127 | 6.6 | Rev1 Polb |
| Rev1 + Polθ | FLAG-siR-Y2387A Polθ | 108 (0) | 0 | 0 | 0 | 108 | 0 | None |
a The number of colonies in which TLS occurred by insertion of a nucleotide other than T is shown in parenthesis.
b The greater reduction in mutation frequency in rows 3 and 5 than in row 1 is because of the increase in TLS frequency that occurs in cells expressing the Y2387A or the Y2387A/Y2391A Polθ (see Table I) and because of the ablation of Polθ-mediated mutagenic TLS by the Y2387A mutation.
c siR, siRNA-resistant.