Table 1.
Functional analysis of MLH3/PMS1 chimera and mlh3 alleles in MMR and meiotic crossing over (CO)
| MMR |
Meiotic CO |
|||||
|---|---|---|---|---|---|---|
| Genotype | Rate × 10−6 (n) | 95% CI. × 10−6 | Relative to WT | % tetratype (n) | Phenotype |
|
| MMR | CO | |||||
| MLH3 | 1.03 (42) | 0.81–1.39 | 1 | 37.1 (1023) | + | + |
| mlh3Δ | 6.24 (39) | 4.53–8.51 | 6.05 | 18.3 (1239) | − | − |
| MLH3-PMS1 chimeras | ||||||
| PPP (PMS1) | 8.29 (15) | 2.85–22.2 | 8.1 | 17.8 (549) | − | − |
| PMM | 15.6 (15) | 3.29–29.0 | 15.1 | 19.2 (530) | − | − |
| MMP | 19.1 (15) | 12.9–25.4 | 18.6* | 21.4 (524) | − − | − |
| PPM | 15.9 (15) | 11.8–128 | 15.4* | 19.8 (824) | − − | − |
| MPP | 15.4 (15) | 12.4–32.3 | 14.9* | 22.3 (837) | − − | − |
| PMP | 17.2 (15) | 13.1–18.2 | 16.7* | 20.7 (1,287) | − − | − |
| MPM | 8.75 (15) | 5.11–11.4 | 8.49 | 21.4 (1,196) | − | − |
| mlh3 Block mutations | ||||||
| Block 1, ATP binding | ||||||
| mlh3-K17T, A20Q, S24D, R30K, Q34D | 2.26 (15) | 1.18–3.78 | 2.20 | 35.6 (513) | +/− | + |
| Block 2, Mlh1 interaction | ||||||
| mlh3-Y493M, N497G, V499F, D500N, K502G | 3.71 (15) | 2.04–4.98 | 3.60 | 29.1 (769) | +/− | +/− |
| mlh3-D500N | 1.73 (15) | 1.33–6.98 | 1.68 | 33.1 (801) | +/− | + |
| mlh3-K502G | 2.84 (15) | 2.13–4.76 | 2.76 | 19.1 (761) | +/− | − |
| Block 3, Endonuclease motif | ||||||
| mlh3-R530K | 2.38 (15) | 1.61–3.28 | 2.31 | 25.3 (771) | +/− | +/− |
| mlh3-R532N | 1.25 (15) | 0.91–2.72 | 1.21 | 29.5 (774) | + | +/− |
| mlh3-R530K, R532N | 4.49 (15) | 3.64–7.24 | 4.36 | 20.4 (509) | − | − |
| Block 4, PCNA interaction motif | ||||||
| mlh3-PIP1 | 3.49 (15) | 1.96–4.35 | 3.39 | 30.9 (742) | − | +/− |
| mlh3-PIP2 | 0.567 (15) | 0.41–0.75 | 0.55 | 37.1 (792) | + | + |
| Block 5, Helix 2 | ||||||
| mlh3-V660K, N666A, F676I, D678K | 4.44 (15) | 2.45–12.8 | 4.32 | 18.2 (760) | − | − |
| mlh3-D678K | 0.831 (15) | 0.57–1.18 | 0.81 | 32.1 (772) | + | +/− |
| Block 6, Helix1 | ||||||
| mlh3-C695L, F699W, A702P, | 3.38 (15) | 2.37–4.76 | 3.28 | 19.5 (527) | +/− | − |
| S707T, V709R, P710H | ||||||
| Mutant combinations | ||||||
| Block 1 (ATP binding), Block 4 (PIP2) | 2.56 (15) | 1.95–4.05 | 2.48 | 31.6 (509) | +/− | +/− |
| Block 5 (Helix 2), Block 6 (Helix 1) | 4.11 (15) | 2.40–5.28 | 3.99 | 17.9 (514) | +/− | − |
| S288C background | ||||||
| wild type + empty vector | 0.28 (15) | 0.19–0.47 | 1.00 | + | ||
| mlh3Δ + empty vector | 1.61 (15) | 1.37–2.08 | 5.90 | − | ||
| mlh3Δ + pMLH3SK1 ARS-CEN | 0.27 (15) | 0.18–0.39 | 0.98 | + | ||
| mlh3Δ + pMLH3SK1-2µ | 223 (15) | 209–291 | 81.8* | − − | ||
The indicated mlh3 Block alleles and chimeras (Supplementary Table S1; Materials and Methods) were integrated into the MLH3 locus in the SK1 strain background and tested for DNA MMR functions using the lys2-A14 reversion assay with 95% CI (confidence interval) presented, and for meiotic crossover functions using a spore-autonomous assay that measures genetic map distances in the CEN8-THR1 interval on Chromosome VIII (Materials and Methods; Supplementary Table S2). For the S288c background experiments, EAY1269 (wild type, S288c background) was transformed with pRS415 (empty vector) and EAY4595 (mlh3Δ) was transformed with pRS415 (empty vector), MLH3SK1-ARS-CEN (pEAA566), and MLH3SK1-2 micron (pEAM65; Supplementary Table S1). n represents the number of independent measurements from at least two transformants. WT, wild type. +, indistinguishable from MLH3 as measured by 95% CI or Chi-Squared (P < 0.0001 for % tetratype). −, indistinguishable from mlh3Δ as measured by 95% CI or Chi-Squared (P < 0.0001). +/− distinguishable from both MLH3 and mlh3Δ as measured by 95% CI or Chi-Squared (P < 0.05). *Mutation rate higher than mlh3Δ; illustrated as a − − phenotype. Note that while expression of MLH3 on an ARS-CEN vector fully complements the mutator phenotype seen in an mlh3Δ strain, expression of MLH3 on a 2µ high copy vector confers a mild mutator phenotype, most likely by sequestering Mlh1 from interacting with Pms1 (Nishant et al. 2008).