Table 1.
Percentages of survival of double strand DNA breaks and mating type switching in the presence of msh3 alleles expressed on low copy plasmids.
| Straina | Plasmidb | % Survivalc | % Switchingd | Tetranucleotide repeat | Summaryf | ||
|---|---|---|---|---|---|---|---|
| Mutation ratee (x 10−6) |
Fold change |
3’NHTR | MMR | ||||
| EAY1042 (MSH3) |
- | 79.8 ± 9.5 | 76.8 ± 6.6 | NDg | - | ||
| EAY1118 (msh3Δ) |
- | 35.4 ± 5.0 | 3.3 ± 3.3 | ND | - | ||
| EAY1118 | MSH3 | 74.2 ±1.7 (100%) |
73.3 ± 2.0 (100%) |
8.6 (7.0–12.5) |
1 | ++ | ++ |
| EAY1118 | Empty Vector | 36.1 ± 2.1 (48.7%) |
7.7 ± 3.9 (10.5%) |
228.5 (179.2–273.7) |
26.5 | − | − |
| Transmitter region: | |||||||
| EAY1118 | msh3R744A | 51.1 ± 3.2 (68.9%) | 13.3 ± 2.9 (18.2%) |
77.9 (65.6–99.5) |
9.0 | − | + |
| EAY1118 | msh3R744L | 33.1 ± 3.4 (44.6%) |
5.0 ± 2.9 (6.8%) | ND | - | − | ND |
| EAY1118 | msh3P745A | 75.6 ± 3.2 (100%) |
44.8 ± 3.8 (61.1%) |
ND | - | + | ND |
| EAY1118 | msh3P745L | 29.7 ± 7.2 (40%) | 1.7 ± 1.7 (2.3%) | ND | - | − | ND |
| EAY1118 | msh3R761A | 80.4 ± 2.9 (100%) |
76.2 ± 4.7(100%) | 9.9 (8.0–14.3) |
1.2 | ++ | ++ |
| EAY1118 | msh3R761L | 67.0 ± 2.6 (90.2%) |
66.6 ± 3.4 (90.9%) |
ND | - | ++ | ND |
| EAY1118 | msh3N762A | 73.5 ± 1.7 (99%) | 71.7 ± 1.7 (97.8%) |
ND | - | ++ | ND |
| EAY1118 | msh3N762L | 54.6 ± 3.5 (73.5%) |
66.6 ± 3.4 (90.9%) |
ND | - | ++ | ND |
| Walker A residues: | |||||||
| EAY1118 | msh3G795A | 43.7 ± 8.7 (58.9%) |
36.7 ± 12.01 (50%) |
145.6 (83.0–250.0) |
16.9 | + | − |
| EAY1118 | msh3G795D | 65.3 ± 4.9 (85.6%) |
50.0 ± 8.1 (65.1%) |
ND | - | + | ND |
| EAY1118 | msh3G796A | 43.0 ± 1.9 (58%) | 22.1 ± 10.0 (30%) |
512.0 (352.4–890.0) |
59.3 | + | − |
| EAY1118 | msh3G796D | 39.0 ± 0.5 (52.5%) |
1.7 ± 1.7 (2.3%) | ND | - | − | ND |
| Aromatic residues: | |||||||
| EAY1118 | msh3P774A | 90.0 ± 4.1 (100%) |
76.7 ± 6.7 (100%) |
9.08 (7.5–11.3) |
1.1 | ++ | ++ |
| EAY1118 | msh3P774L | 75.6 ± 6.2 (100%) |
66.7 ± 1.7(91%) | ND | - | ++ | ND |
| EAY1118 | msh3Y925A | 64.7 ± 12 (87.2%) |
61.7 ± 6.0 (84.2%) |
312.7 (290.6–338.6) |
36.3 | ++ | − |
| EAY1118 | msh3F940A | 45.2 ± 3.8 (60.9%) |
30.0 ± 4.3 (40.9%) |
196.0 (117.2–299.5) |
22.7 | + | − |
| EAY1118 | msh3Y942A | 51.3 ± 4.7 (69.1%) |
61.7 ± 4.4 (84.2%) |
120.0 (98.8–169.2) |
13.9 | ++ | − |
Strains carrying a double non-homology at the mating type locus were used to determine Msh2-Msh3-dependent 3’NHTR. Genotypes of the strains are provided in Table S1.
EAY1118 was transformed with low copy plasmids bearing MSH3 or msh3 alleles or with the empty vector to determine 3’NHTR. The plasmids are listed in Table S3. The strain numbers are listed in Table S4.
Percentage cell survival (induced/uninduced) was determined by examining the viability of cells plated after a 45 minute induction of HO expression. The standard error of the mean is indicated. The percentage in parentheses indicated the activity of each allele relative to the activity in the presence of the MSH3 plasmid.
Surviving cells were assayed to determine the percentage that had switched mating type following HO expression and had therefore undergone DSBR (see Materials and Methods). The percentage in parentheses indicated the activity of each allele relative to the activity in the presence of the MSH3 plasmid.
EAY1118 carrying empty vector or MSH3 low copy plasmids was transformed with the tetranucleotide repeat reporter plasmid (pBK1). Mutation rates were determined as described in Materials and Methods. Numbers in parentheses represent 95% confidence intervals.
Each allele was characterized as having wild type function (>90% activity; ++), intermediate phenotype (20–90% activity; +) or null phenotype (<20% activity;-) in either 3’ NHTR or MMR.
ND = Not Determined