Table 4. MLH1 missense mutations identified in human-yeast hybrid MLH1_h(77-134).
| MLH1 gene or variant codon # | Screening methoda | Missense mutation | Consequence | Corresponding human residue | Mutation defectb | Number of times isolated |
|---|---|---|---|---|---|---|
| Yeast codon | ||||||
| 30 | a | AAA→AAT | K30N | K33 | +++ | 1 |
| 35 | a | AAT→AGT | N35S | N38 | +++ | 1 |
| 37 | a | ATC→TTC | I37F | L40 | ++ | 1 |
| a | ATC→ACC | I37T | L40 | +++ | 1 | |
| 38 | a,a,b | GAT→GGT | D38G | D41 | +++ | 3 |
| b | GAT→GAA | D38E | D41 | +++ | 1 | |
| b | GAT→ATT | D38N | D41 | +++ | 1 | |
| 40 | b | AAT→ATT | N40Ic | K43 | +++ | 1 |
| 41 | a,a | GCT→GTT | A41V | S44 | ++ | 2 |
| 42 | a | ACA→ATA | T42I | T45 | +++ | 1 |
| 45 | b | GAT→GGT | D45G | Q48 | + | 1 |
| 46 | b | ATT→AAT | I46N | V49 | +++ | 1 |
| 49 | a | AAG→GAG | K49E | K52 | ++ | 1 |
| 50 | a | GAA→GTA | E50V | E53 | + | 1 |
| 52 | a,a | GGA→AGA | G52R | G55 | + | 2 |
| 56 | b | CTT→CAT | L56H | I59 | +++ | 1 |
| 58 | a,b | ATA→AAA | I58K | I61 | +++ | 2 |
| 60 | a | GAT→GGT | D60G | D63 | ++ | 1 |
| 61 | b | AAC→AGC | N61S | N64 | +++ | 1 |
| 62 | a,b | GGA→GAA | G62E | G65 | +++ | 2 |
| a,a | GGA→AGA | G62R | G65 | ++ | 2 | |
| 65 | a | ATT→AAT | I65N | I68 | +++ | 1 |
| 71 | a | CCA→CTA | P71L | D74 | ++ | 1 |
| Human codon | ||||||
| 77 | a | TGT→CGT | C77R | — | ++ | 1 |
| 78 | a | GAG→GTG | E78V | — | ++ | 1 |
| 80 | a,a | TTC→CTCd | F80Lc | — | +++ | 2 |
| 89 | a | GAG→GTG | E89V | — | + | 1 |
| 99 | a | TTT→ATT | F99I | — | +++ | 1 |
| 99 | a | TTT→CTT | F99L | — | ++ | 1 |
| 100 | b | CGA→CAA | R100Q | — | ++ | 1 |
| 101 | a | GGT→GAT | G101Dc | — | +++ | 1 |
| 103 | a | GCT→GTT | A103V | — | ++ | 1 |
| a,b | GCT→ACT | A103T | — | ++ | 2 | |
| a | GCT→CCT | A103P | — | ++ | 1 | |
| 111 | a | GCT→ACT | A111T | — | +++ | 1 |
| 114 | a | ACT→ATT | T114I | — | ++ | 1 |
| 115 | b | ATT→AGT | I115Sc | — | +++ | 1 |
| b | ATT→AAT | I115N | — | +++ | 1 | |
| b | ATT→TTT | I115F | — | ++ | 1 | |
| 116 | a | ACA→TCA | T116S | — | + | 1 |
| 118 | b | AAA→AAT | K118N | — | +++ | 1 |
| a | AAA→ATA | K118I | — | + | 1 | |
| 133 | a | GGA→GAA | G133E | — | ++ | 1 |
| Yeast codon | ||||||
| 136 | a | CCC→CAC | P136H | P139 | + | 1 |
| 140 | a | GCT→GTT | A140V | A143 | ++ | 1 |
| 144 | a | GGT→AGT | G144S | G147 | ++ | 1 |
aMMR-deficient transformants were identified by (Method ‘a’) qualitative patch assays using YBT24 or (Method ‘b’) colorimetric assay using YBT41 as described in the Materials and Methods.
bYeast strain YBT24 containing pSH91 was transformed with pMLH1_h(77-134) containing the indicated missense mutations. Mutation frequencies were determined using a standardized MMR assay based on instability of the GT-tract in pSH91 [Ellison et al., (51)]. To calculate the mutation defect, the mean mutation frequency conferred by each variant was divided by the mutation frequency conferred by the parental MLH1_h(77-134) gene. +, Mutation defect of 2.5–9.0 (9–33% loss-of-MMR function relative to the mutation frequency of the MLH1-null strain YBT24); ++, Mutation defect of 9.1–17.9 (34–66% loss-of-MMR function); +++, Mutation defect of 18.0 or greater (>67% loss-of-MMR function). The mean mutation frequency conferred by pMLH1_h(77-134) was 1.2 × 10−4 (range: 0.6–2.4 × 10−4). The mean mutation frequency conferred by the empty expression vector pMETc was 3.3 × 10−3 (range: 1.8–7.0 × 10−3) (mutation defect = 27.5).
cIn addition to the indicated missense mutation the following silent alterations were observed (mutation/silent alteration): N40I/K134K; F80L/A92A; G101D/K54K; I115S/T116T.
dThe missense mutation TTC→TTA was also identified.