Table 3. MLH1 missense mutations identified in human-yeast hybrid MLH1_h(41-86).

MLH1 gene or variant codon #	Screening methoda	Missense mutation	Consequence	Corresponding human residue	Mutation defectb	Number of times isolated
Yeast codon
8	a	CTT→CAC	L8H	L11	++	1
16	a	ATT→TTT	I16F	I19	++	1
26	a	GTA→ATA	V26I	A29	++	1
35	a	AAT→GAT	N35D	N38	+++	1
	a	AAT→ACT	N35T	N38	++	1
37	a,a	ATC→ACC	I37T	L40	+++	2
	b	ATC→AAC	I37N	L40	+++	1
Human codon
41	a	GAT→GGT	D41G	—	++	1
42	a	GCA→ACA	A42T	—	+++	1
	b	GCA→GAA	A42E	—	+++	1
	b	GCA→GTA	A42Vc	—	+++	1
44	a	TCC→TTC	S44F	—	+++	1
45	a,b	ACA→ATA	T45I	—	+++	2
46	a	AGT→ACT	S46T	—	++	1
47	b	ATT→ACT	I47T	—	++	1
	a	ATT→AGT	I47S	—	+++	1
48	a	CAA→TAT	Q48Y	—	+++	1
49	b	GTG→GAG	V49E	—	++	1
	a	GTG→ATG	V49M	—	++	1
	a	GTG→GCG	V49A	—	+++	1
51	a,b	GTT→GAT	V51D	—	++	2
	a	GTT→GCT	V51A	—	++	1
52	a,a,b	AAA→ATA	K52I	—	+	3
53	a,a,b	GAG→GTG	E53V	—	++	3
54	a	GGA→AGA	G54R	—	+	1
55	a,a,b	GGC→GAC	G55D	—	+	3
	a	GGC→AGC	G55S	—	++	1
56	a	CTG→ATG	L56M	—	+	1
	a	CTG→CCG	L56P	—	+++	1
57	a	AAG→GAG	K57Ec	—	+	1
	b	AAG→AAC	K57N	—	+++	1
59	b	ATT→AAT	I59N	—	+++	1
	a,a	ATT→TTT	I59F	—	+++	2
	a	ATT→ACT	I59T	—	+++	1
60	a	CAG→CCG	Q60P	—	++	1
61	a	ATC→AAC	I61N	—	++	1
63	a	GAC→TAC	D63Y	—	+++	1
64	b	AAT→ATT	N64I	—	++	1
65	b	GGC→GTC	G65V	—	+++	1
	a	GGC→GCC	G65A	—	+++	1
	a	GGC→GAC	G65D	—	++	1
	a	GGC→AGC	G65S	—	++	1
67	a,a,b	GGG→GAG	G67E	—	++	3
	a	GGG→GTG	G67V	—	+++	1
68	a	ATC→AAC	I68N	—	+++	1
	a,b	ATC→TTC	I68F	—	++	2
	b	ATC→AGC	I68Sc	—	++	1
70	a	AAA→AAT	K70N	—	+++	1
	a	AAA→ATA	K70I	—	+++	1
72	a,b	GAT→GGT	D72G	—	++	2
	a,b	GAT→GTT	D72V	—	+	2
73	b	CTG→ATG	L73M	—	++	1
	a	CTG→CCG	L73P	—	++	1
	a	CTG→CAG	L73Q	—	++	1
76	b	GTA→GAA	V76E	—	+++	1
77	a	TGT→GGT	C77G	—	++	1
	b	TGT→TCT	C77S	—	++	1
79	b	AGG→TGG	R79Wc	—	++	1
80	a	TTC→TCC	F80Sc	—	++	1
	b	TTC→CTC	F80L	—	+++	1
	b	TTC→ATC	F80I	—	+++	1
81	a	ACG→ATG	T81M	—	+	1
82	a,a	ACG→TCG	T82S	—	+	2
	a,b	ACG→AAG	T82K	—	+++	2
	a	ACG→ATG	T82M	—	+++	1
83	a,b	TCC→CCC	S83P	—	++	2
	a	TCC→TTC	S83F	—	+++	1
84	a	AAA→GAA	K84R	—	++	1
	a	AAA→AGA	K84E	—	++	1
85	a	TTA→TCA	L85S	—	++	1
Yeast codon
86	a	GAA→GGA	E86Gc	E89	++	1
88	a	TTG→GTG	L88V	L91	++	1
99	b	GAA→GGA	E99G	E102	++	1
108	a	GCA→CCA	A108P	A111	+++	1
110	a	GTC→GCC	V110Ac	V113	+++	1
112	b	GTA→GAA	V112E	I115	++	1
113	b	ACG→GCG	T113A	T116	++	1
144	a	GGT→AGT	G144S	G147	+++	1

^aMMR-deficient transformants were identified by (‘a’) qualitative patch assays using YBT24 or (‘b’) colorimetric assay using YBT41 as described in the Materials and Methods section.

^bYeast strain YBT24 containing pSH91 was transformed with pMLH1_h(41-86) 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(41-86) gene. +, Mutation defect of 2.1 to 3.9 (18–33% loss-of-MMR function relative to the mutation frequency of the MLH1-null strain YBT24); ++, Mutation defect of 4.0–7.6 (34–66% loss-of-MMR function); +++, Mutation defect of 7.8 or greater (>67% loss-of-MMR function). The mean mutation frequency conferred by pMLH1_h(41-86) was 2.7 × 10⁻⁴ (range: 1.1–4.4 × 10⁻⁴) The mean mutation frequency conferred by the empty expression vector pMETc was 3.2 × 10⁻³ (range: 1.9–7.0 × 10⁻³) (mutation defect = 11.7).

^cIn addition to the indicated missense mutation, the following silent alterations were observed (mutation/silent alteration): A42V/F85F; K57E/T45T; I68S/I47I and I75I; R79W/D143D; F80S/L73L; E86G/T82T and K142K; V110A/T66T.