Table 2.
mlh3-ATP binding and mlh3-PIP mutations modestly rescue pms1 null MMR defects
| pms1Δ strain with indicated plasmids | Rate × 10−7 (n) | 95% CI × 10−7 | Relative to wild type |
|---|---|---|---|
| PMS1, ARS-CEN | 2.99 (30) | 2.11–3.85 | 1 |
| PMS1, 2µ | 22.4 (18) | 15.1–63.5 | 7.50 |
| ARS-CEN (LEU2) | 20,300 (29) | 12,900–28,700 | 6,800 |
| 2µ | 18,900 (18) | 15,900–21,300 | 6,320 |
| mlh3 Block mutations, ARS-CEN | |||
| MLH3 | 14,700 (15) | 8,620–19,700 | 4,910 |
| Block 4 (mlh3-PIP1) | 14,400 (15) | 6,470–18,600 | 4,830 |
| Block 4 (mlh3-PIP2) | 11,300 (15) | 7,890–21,200 | 3,790 |
| mlh3 Block mutations, 2µ | |||
| Block 1, ATP binding | |||
| mlh3-K17T, A20Q, S24D, R30K, Q34D | 9,720 (18) | 6,360–11,300 | 3,250* |
| Block 2, Mlh1 interaction | |||
| mlh3-Y493M, N497G, V499F, D500N, K502G | 15,400 (18) | 10,700–23,700 | 5,160 |
| mlh3-D500N | 10,900 (18) | 9,540–16,100 | 3,630 |
| mlh3-K502G | 16,500 (18) | 10,200–19,100 | 5,530 |
| Block 3, Endonuclease motif | |||
| mlh3-R530K | 20,500 (18) | 16,900–28,600 | 6,840 |
| mlh3-R532N | 15,700 (18) | 10,600–21,600 | 5,240 |
| mlh3-R530K, R532N | 13,300 (18) | 10,100–17,200 | 4,440 |
| Block 4, PCNA interaction motif | |||
| MLH3 (GTFVAR) | 13,200 (28) | 8,280–20,600 | 4,430 |
| mlh3-PIP1 (QKLIIP) | 13,700 (25) | 7,940–17,200 | 4,590 |
| mlh3-PIP2 (QTFIAP) | 6,300 (44) | 4,120–7,470 | 2,110* |
| mlh3-PIP3 (QTLIAP) | 8,520 (18) | 6,950–17,600 | 2,850 |
| mlh3-PIP4 (GTFIAP) | 13,800 (18) | 7,390–22,400 | 4,610 |
| mlh3-PIP5 (QTFIAR) | 6,890 (18) | 4,250–9,890 | 2,300* |
| mlh3-PIP6 (QTFVAP) | 15,700 (18) | 13,100–25,800 | 5,240 |
| mlh3-PIP7 (GTFVAP) | 15,500 (18) | 10,500–22,500 | 5,190 |
| mlh3-PIP8 (GTFIAR) | 19,500 (18) | 15,100–25,900 | 6,510 |
| mlh3-PIP9 (QTFVAR) | 8,520 (18) | 4,890–10,800 | 3,060* |
| Block 5, Helix 2 | |||
| mlh3- V660K, N666A, F676I, D678K | 14,900 (18) | 10,700–19,700 | 4,970 |
| mlh3-D678K | 14,700 (18) | 11,900–22,200 | 4,920 |
| Block 6, Helix 1 | |||
| mlh3-C695L, F699W, A702P, S707T, V709R, P710H | 14,100 (18) | 12,100–21,100 | 4,710 |
| Double mutants | |||
| Block 1, Block 4 (PIP2) | 5,480 (18) | 4,340-6,710 | 1,830* |
| Block 2 (D500N), Block 4 (PIP2) | 12,200 (18) | 6,340–15,100 | 4,078 |
| Block 2 (K502G), Block 4 (PIP2) | 11,900 (18) | 6,830–19,100 | 3,970 |
| Block 3 (R530K), Block 4 (PIP2) | 7,350 (18) | 2,490–12,700 | 2,460* |
| Block 3 (R532N), Block 4 (PIP2) | 7,110 (18) | 3,240–9,650 | 2,380* |
| Block 3 (R530K, R532N), Block 4 (PIP2) | 17,600 (18) | 13,400–19,800 | 5,880 |
| Block 4 (PIP2), Block 5 (D678K) | 11,000 (18) | 5,750–12,900 | 3,690* |
| Block 4 (PIP2), Block 5 (Helix 2) | 18,700 (18) | 12,200–30,900 | 6,240 |
| Block 4 (PIP2), Block 6 (Helix 1) | 20,600 (18) | 14,400–27,900 | 6,904 |
| Block 5 (Helix 2), Block 6 (Helix 1) | 20,200 (18) | 17,500–38,800 | 6,740 |
| MLH3-PMS1 chimeras, 2µ | |||
| MMM | 13,200 (28) | 8,280–20,600 | 4,430 |
| PPP | 42.9 (18) | 24.1–91.4 | 14.4 |
| PMM | 19,600 (18) | 8,530–31,200 | 6,550 |
| MMP | 14,600 (18) | 11,600–21,100 | 4,870 |
| PPM | 16,100 (18) | 12,900–18,500 | 5,380 |
| MPP | 14,800 (18) | 11,800–18,300 | 4,960 |
| PMP | 15,300 (18) | 12,800–21,300 | 5,130 |
| MPM | 20,400 (18) | 18,100–35,100 | 6,830 |
EAY3097 (pms1Δ, S288c background) was transformed with pJH481 (PMS1S288c, ARS-CEN), pEAM50 (PMS1S288c, 2µ), and pRS415 (dummy vector; Supplementary Table S1) to analyze PMS1, PMS1-2µ, and pms1Δ genotypes, respectively. mlh3 substitution alleles (Blocks 1–6, Figure 2; Supplementary Table S1) and MLH3-PMS1 chimera constructs (Supplementary Table S1) were also transformed into EAY3097. mlh3 alleles and chimeras were expressed from an MLH3 promoter in 2µ and ARS-CEN vectors as indicated. All strains were analyzed for mutation rate using the lys2-A14 reversion assay as described in the Materials and Methods with the 95% confidence interval (CI) presented. n represents the number of independent measurements obtained from at least two transformants. *Indicates complementation of pms1Δ as measured by nonoverlap in 95% CI.