Table 7.
Rad18 is involved in gap-lesion repair by TLS but not in HDR
| Cell line |
E. coli transformantsa |
Plasmid repair (%) | TLSb (%) | HDRb (%) | |
| KanR | CmR | ||||
| Rad18−/− | 75 | 320 | 23.8 ± 10.6 | <1.2 ± 0.5 | 22.6 ± 10 |
| Rad18+/+ | 78 | 140 | 58.4 ± 9 | 21.6 ± 3.3 | 27.4 ± 4.3 |
| Number of isolates (%) |
||
| Cell line | Rad18−/− | Rad18+/+ |
| Plasmid type | GP-BPG + hDNA | GP-BPG + hDNA |
| Event type | ||
| Base substitutions | ||
| A | – | 2 |
| C | – | 4 |
| G | – | 1 |
| T | – | – |
| (−1) | – | – |
| Total TLS | 0 (<5%) | 7 (37%) |
| Control GP sequence | 1 | 1 |
| HDR product | 18 (95%) | 9 (47%) |
| Other eventsc | – | 2 |
| Total number of isolates | 19 | 19 |
Rad8−/− and Rad18+/+ MEF cells were transfected with DNA mixture containing the gap plasmid bearing a BP-G adduct (kanR) and the control gap-plasmid (cmR) in the presence of hDNA. Following 10 h incubation, the DNA was extracted and used to transform an E. coli indicator strain. Plasmid repair was calculated based on the ratio of kanR/cmR colonies. Each result represents the average of at least four experiments. Results of single gap-filling events were obtained by sequence analysis of plasmid DNA extracted from single kanR colonies. Depicted is the sequence at the position across from the lesion.
aThe number of transformants obtained in a typical assay with 100 μl of transformation mixture.
bThe repair by HDR or TLS was calculated by multiplying total plasmid repair levels by the fraction of HDR or TLS events out of the total sequences analyzed.
cOther events depict several base pair deletions or insertion of non homologous sequences.