Table 1. Summary of the in vitro and in vivo characterization of PCNA mutants.
| PCNA (POL30) Mutant | IDCL Sequencea | Binding of PCNA Mutants to Target Partnersb | Spontaneous Mutation Ratee CanR (Fold Increase) | ||||
| Rad30 | Rad27 | Pol32 | Msh6 | Ung1 | |||
| POL30 | DIDADFLKIEEL | 1 | 1 | 1 | 1 | 1 | 1.5 [1.5, 2.2]×10−7 |
| pol30-Rad30E2 | DDDWDFLKILEL | 1.4 c | 0.4 c | 0.4 c | 1 | 1 | 1.5 [1.5, 2.0]×10−6 (10) |
| pol30-Rad30E9 | DNDWDFLKIDEL | 1.6 c (8.5) d | 0.5 c | 1 | 1 | 1 | 2.3 [1.7, 3.0]×10−6 (15.3) |
| pol30-Rad27E6 | DGDYDILKIREL | 1 | 1.5 c | 1 | 1 | 1 | 1.2 [0.8, 1.4]×10−6 (8) |
| pol30-Rad27E31 | DGDYDVLKIREL | 1 | 1.5 c | 1 | 1 | 1 | ND |
| pol30-Rad27L1f | D––VDILKIGEL | 0.4 | 1.7 c | 1 | 1 | 1 | NA |
| pol30-Rad27L2f | D––VDTLKITEL | 0.5 | 1.5 c | 1 | 1 | 1 | NA |
| pol30-Pol32E5 | DADNDFLKISEL | 1 | 1 | 1.5 c | 1 | 1 | 2.0 [1.4, 2.8]×10−6 (13.3) |
| pol30-Pol32E9 | DNDVDSLKIIEL | 1 | 1.2 | 1.3 c (2) d | 1 | 1 | 6.0 [4.7, 7.1]×10−7 (4) |
| pol30-Msh6E2 | DYDRDMLKISEL | 1 | 1.3 | 1 | 1.4 c | 1 | 1.1 [0.7, 1.6]×10−6 (7.3) |
| pol30-Msh6E6 | DYDKDLLKIIEL | 1 | 1.3 | 1 | 1.4 c (1.6) d | 1 | 6.1 [5.1, 8.8]×10−7 (4.1) |
| pol30-Ung1E2 | DDDSDFLKIPEL | 1 | 1 | 1 | 1 | 1.3 c | 3.3 [2.2, 3.3]×10−7 (2.2) |
| pol30-Ung1E3 | DFDYDELKIEEL | 1 | 1 | 1 | 1 | 1.3 c (3.5) d | 3.1 [2.1, 3.9]×10−7 (2.1) |
| pol30-79 | DIDADFAKAEEL | 0.4 c | 0.5 c | 0.3 c | 0.4 c | 0.3 c | 3.3 [2.2, 5.1]×10−7 (2.2) |
The IDCL sequences of the PCNA mutants. The positions that were diversified in the naïve library and mutations identified in the selected mutants are marked in bold. The mutations in pol30-79 [31] are marked in bold.
Binding characterization of the PCNA mutants for the five target partners (see Figure 2) using Y2H and SPR assays. Growth of the Y2H strain containing WT and mutant PCNA and the different partners was analyzed on selective agar plates lacking histidine. Growth rates similar to that of cells expressing WT PCNA are denoted as 1. Increase and decrease in affinity detected by Y2H or SPR assays are marked in bold.
Fold increase or decrease in the PCNA-partner interaction affinities measured as the generation time of the Y2H strains grown in liquid selective media lacking histidine, relative to cells expressing WT PCNA (see Figures S4 and Materials and Methods).
Fold increase in binding affinities of PCNA mutants for PIP peptides derived from the different partners (in parenthesis), relative to WT PCNA, as measured by SPR analysis. Binding affinities of the WT PCNA for Rad30, Pol32, Msh6, and Ung1 PIP peptides are 1.7×10−7M, 3.6×10−7M, 3×10−6M, and 1.2×10−6M, respectively. The off-rate (kd) of the Rad30 PIP peptide measured for the WT and pol30-Rad30E9 mutant are 0.03 s−1 and 0.00321 s−1, respectively.
The spontaneous mutation rate was determined by fluctuation analysis (see Materials and Methods). Values in brackets represent the low and high limits for the 95% confidence interval obtained for each rate. The numbers in parentheses indicate the fold increase, as compared to the WT POL30 strain. The differences between the mutation rate of the mutants and the WT are significant (p<0.002 in all cases, Mann-Whitney test).
Strains containing these mutants as the sole source of PCNA are non-viable.
ND, not determined; NA, not applicable.