Table 4.
Effects of Y2387A, Y2391A, or Y2387A/Y2391A mutations in Polθ on mutation frequency and nucleotides inserted opposite ϵdA carried on the leading-strand DNA template in Polθ−/− MEFs
| MEFs (siRNA) | Vector expressing | No. of Kan+ blue colonies sequenced | Nucleotide inserted |
Mutation frequency | Error-prone pathway that remains active | |||
|---|---|---|---|---|---|---|---|---|
| A | G | C | T | |||||
| % | ||||||||
| Polθ−/− | Vector control | 144 (15)a | 3 | 1 | 11 | 129 | 10.4 | Rev1Pol |
| Polθ−/− | WT Polθ | 138 (20) | 5 | 1 | 14 | 118 | 14.5 | Rev1Pol, Polθ |
| Polθ−/− | Y2387A Polθ | 140 (11) | 2 | 0 | 9 | 129 | 7.9 | Rev1 Pol |
| Polθ−/− | Y2391A Polθ | 144 (40) | 13 | 5 | 22 | 104 | 27.8 | Rev1 Pol, Y2391A Polθ |
| Polθ−/− | Y2387A/Y2391A Polθ | 152 (11) | 2 | 0 | 9 | 141 | 7.2 | Rev1 Pol |
| Polθ−/− (mRev1 siRNA)b | WT Polθ | 96 (7) | 4 | 0 | 3 | 89 | 7.3 | Polθ |
| Polθ−/− (mRev1 siRNA) | Y2387A Polθ | 96 (0) | 0 | 0 | 0 | 96 | 0.0 | None |
| Polθ−/− (mRev1 siRNA) | Y2391A Polθ | 90 (18) | 8 | 4 | 6 | 72 | 20.0 | Y2391A Polθ |
| Polθ−/− (mRev1 siRNA) | Y2387A/Y2391A Polθ | 104 (0) | 0 | 0 | 0 | 104 | 0.0 | None |
a The number of colonies in which TLS occurred by insertion of a nucleotide other than T is shown in parenthesis.
b mRev1, mouse Rev1 siRNA.