Functional mapping of the fission yeast DNA polymerase δ B-subunit Cdc1 by site-directed and random pentapeptide insertion mutagenesis

Table 1 Pentapeptide insertion and site-directed mutants: a summary

Mutant	Sub-domain	Location	Inserted sequence or point mutation(s)	Ability to substitute for Cdc1 at different expression levels		Interaction with Pol3 and Cdc27 by two-hybrid analysis (%)
				Low	High	Pol3	Cdc27
WT				+	+	100	100
J12	N	7	Ser-GlyValProHisSer-Ile	+	+	<1	59
E4	N	10	Cys-GlyValProProCys-Glu	+	+	16	62
J2	I	25	Tyr-ArgGlyThrProTyr-Ser	+	+	46	<1
E2	I	26	Ser-GlyValProLysTer	-	-	<1	<1
J17	I	28	Gln-Ter	-	+	2	<1
J3	I/II	62	Leu-GlyValProLeuLeu-Asp	-	+	54	<1
E1	I/II	66	Ser-GlyValProGlnSer-Asp	+	+	65	1
J10	II	77	Tyr-ArgGlyThrProTyr-Met	-	+	<1	30
J8	II	83	Lys-GlyValProLeuLys-Pro	+	+	<1	128
E5	II	86	Val-MetGlyTyrProVal-Met	+	+	48	88
J6	III	124	Tyr-GlyValProHisTyr-Gly	-	+	<1	<1
E9	III	124	Tyr-GlyGlyTyrProTyr-Gly	-	+	24	<1
J4	III	127	Ile-GlyValProArgIle-Asp	-	+	36	<1
J1	III	141	Thr-GlyValProLeuThr-Gly	-	-	<1	<1
J16	III/IV	160	Val-GlyGlyThrProVal-Asp	-	-	<1	<1
J9	III/IV	174	Met-ArgGlyThrProMet-Thr	+	+	91	102
J15	IV/V	203	Gln-GlyValProLeuGln-Val	+	+	1	78
J22	IV/V	212	Arg-GlyValProLeuArg-Gly	-	+	25	7
J21	V/VI	275	Leu-GlyValProGlnLeu-Asp	-	+	<1	<1
A1	VI	292	Pro292Ala	+	+	39	44
A2	VI	293	Gly293Ala	+	+	<1	19
A9	VI	292–293	Pro292Ala, Gly293Ala	-	-	<1	58
A3	VI	296	Asp296Ala	-	-	<1	23
A4	VI	303	Pro303Ala	+	+	<1	31
A5	VII	328	Asn328Ala	+	+	4	37
A6	VII	329	Pro329Ala	-	+	<1	6
A10	VII	328–329	Asn328Ala, Pro329Ala	-	-	<1	72
A7	VII	344	Gly344Ala	+	+	9	<1
A8	VIII	374	Pro374Ala	+	+	17	8
J7	VIII	393	Met-GlyValProLeuMet-Glu	-	-	<1	19

For the pentapeptide insertion mutants, multiply isolated insertions were as follows: J2 (re-isolated as J18, J20, E10), J3 (E3, E8), J10 (J11, E6, E7), J8 (J19) and J4 (J5, J13). The location of the insertions is given as the number of the last amino acid residue N-terminal to the insertion. The insertion sequences are shown between the hyphens. Note that the target site duplication results in the amino acid immediately N-terminal to the insertion being duplicated as the fifth amino acid of the insertion (except where stop codons are inserted) and that the insertions in Cdc1-J6 and Cdc1-E9 are found at the same amino acid but are of different sequence. Quantitative two-hybrid assay values are given as percentages of the interaction seen with wild-type Cdc1 with Pol3 (in the presence of Cdm1) or Cdc27. See Results for further details.

ISSN: 1471-2199