Table 1.
DNA sequence analysis of TLS events across BP-G and cisPt-GG in human U2OS cells, in which the expression of specific TLS DNA polymerases was knocked down using siRNA
| siRNA | Control (%) | REV3L (%) | POLK (%) | POLH (%) | POLK +REV3L (%) | POLK+POLH (%) |
|---|---|---|---|---|---|---|
| Nucleotide inserted opposite BP-G | Number of isolates | |||||
| C | 127 (74.3) | 52 (36.1) | 76 (68) | 65(69.9) | 55 (42.3) | 83 (60.1) |
| A | 11 (6.4) | 5 (4.5) | 3 (3.2) | 2 (1.4) | ||
| G | 6 (3.5) | 2 (1.4) | 2 (1.5) | 1 (0.7) | ||
| T | 3 (1.8) | 2 (1.4) | 2 (1.8) | 1 (0.7) | ||
| Deletion/insertion | 24 (14) | 88 (61.1) | 29 (25.9) | 25 (26.9) | 73 (56.2) | 51 (37) |
| Total number | 171 (100) | 144 (100) | 112 (100) | 93 (100) | 130 (100) | 138 (100) |
| TLS clonesa | 147 | 56 | 83 | 68 | 57 | 87 |
| Mutagenic TLS (%)b | 13.6 | 7.1 | 8.4 | 4.4 | 3.5 | 4.6 |
| P=0.2c | P=0.2c | P=0.042c | P=0.037c | P=0.03c | ||
| Nucleotide inserted opposite cisPt-GG | Number of isolates | |||||
| CC | 98 (78.4) | 53 (53) | 60 (87) | 59 (70.2) | 50 (58.1) | 44 (77.2) |
| AC | 15 (12) | 9 (9) | 2 (2.9) | 7 (8.3) | 4 (4.7) | 1 (1.8) |
| TC | 6 (4.8) | 3 (3.6) | ||||
| CT | 1 (0.8) | |||||
| CA | 2 (2.3) | |||||
| Deletion/insertion | 5 (4) | 38 (38) | 7 (10.1) | 15 (17.9) | 30 (34.9) | 12 (21.1) |
| Total isolates | 125 (100) | 100 (100) | 69 (100) | 84 (100) | 86 (100) | 57 (100) |
| TLS clonesa | 120 | 62 | 62 | 69 | 56 | 45 |
| Mutagenic TLS (%)b | 18.3 | 14.5 | 3.2 | 14.5 | 10.7 | 2.2 |
| P=0.006c | P=0.01c | |||||
| aThe number of clones, excluding clones having large deletions or insertions. | ||||||
| bThe percentage of mutagenic TLS is the fraction of events other than insertion of C opposite BP-G, or CC opposite cisPt-GG out of the total number of TLS events. | ||||||
| cP-value is given compared with the results obtained with cells transfected with control siRNA. Analysis was performed using the χ2 test. P-values that reached statistical significance are in bold face type. | ||||||
| U2OS cells were transfected with the indicated siRNAs. After incubation of 72 h, the plasmid mixtures containing the gap-lesion plasmid GP-BP-G1 or GP-cisPt-GG (kanR), along with the control gapped plasmid GP20 (cmR) and the carrier plasmid were introduced into the cells. Following incubation of 8 h to allow TLS, the DNA was extracted and used to transform a recA E. coli indicator strain. GP-BP-G1 or GP-cisPt-GG (kanR) descendents were extracted from kanR colonies and subjected to DNA sequence analysis. Deletions and insertions are taken as non-TLS events. | ||||||