Nucleic Acids Research, Vol. 20, No. 2 375
Nucleotide sequence of the POL3 gene encoding DNA polymerase III (6) of Saccharomyces cerevisiae Alan Morrison and Akio Sugino Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA Submitted September 16, 1991 We have described mutational studies of the conserved 3'- 5' exonuclease domain of S. cerevisiae DNA polymerase II (e) (1). On extending these studies to S. cerevisiae DNA polymerase III (6) in experiments similar to those now reported by Simon et al. (2), we noticed several differences between the nucleotide sequence of part of the POL3 gene (formerly CDC2) determined by us and the original sequence of Boulet et al. (3). Some of these differences were outside of the BspEI-BspEI fragment (nucleotides 973-1350) used for site-directed mutagenesis, and therefore had not arisen during this procedure. Neither was polymorphism the likely explanation, since we were using the POL3 plasmid pBL304, constructed in P.M.J.Burgers' laboratory and derived from pAB15 described by Boulet et al. (3). We considered whether these differences might have been mutations that had appeared during propagation of plasmid DNA in E. coli since the POL3 gene renders plasmids unstable. One of the nucleotide sequence differences, G923-A, removed a ThaI cleavage sequence present in the original sequence. We observed that ThaI did not cut at this site either in pBL304 or in yeast chromosomal DNA amplified by the polymerase chain reaction. Subsequently, we determined the nucleotide sequence of the entire POL3 gene of pBL304 by sequencing double stranded DNA using synthetic oligonucleotide primers and Sequenase (United States Biochemical Corporation). In addition we sequenced an independently isolated POL3 clone, HL1, provided by D.A.Gordenin. HL1 was isolated from a plasmid library of yeast genomic DNA by complementation of the tex] allele of POL3 (4). These two POL3 nucleotide sequences were identical. Table I summarizes the differences between the original and new sequences. The new nucleotide sequence begins at the MluI site and extends for 3551 nucleotides, corresponding to positions 193-3730 of the original sequence. The DNA polymerase III polypeptide predicted by the new sequence is 1097 amino acids long and differs from that described in reference 3 in twenty-six amino acid positions. None of the changes, however, occurs within the conserved motifs of the 3'-5' exonuclease or
EMBL accession no. X61920 polymerase domains. The biggest difference is in a sequence of seventeen residues (347 -363) occurring between the 'ExoI' and 'ExoHI motifs (1, 2).
REFERENCES 1. Morrison,A., Bell,J.B., Kunkel,T.A. and Sugino,A. (1991) Proc. Natl. Acad. Sci. USA 88, 9473-9477. 2. Simon,M., Giot,L. and Faye,G. (1991) EMBO J. 10, 2165-2170. 3. Boulet,A., Simon,M., Faye,G., Bauer,G.A. and Burgers,P.M.J. (1989) EMBO J. 8, 1849-1854. 4. Gordenin,D.A., Malkova,A.L., Peterzen,A., Kulikov,V.N., Pavlov,Y.I., Perkins,E. and Resnick,M.A. (1991) Proc. Natl. Acad. Sci. USA 88 (in press).
Table 1. Changes to the PKX3 sequence. I
S uoe ---de New positton Charge
Amino Acid Se_afe New positn Change 0
339
147
+T
Old position -
-
-
591
400
C -G
78
78
D-E
Old positon
592
401
G -C
79
79
V-+L
923
732
G -A
189
189
R-+H
968
777
A-+G
204
204
D -G
1059
868
A-T
234
234
-
1396 1399 1433 1440
1205 -6
+ AG
347 - 361
347 - 363
1210-11 1246 1254
YLALRNHSF VMCYSD -.
+A
1965
1780
T-+A
536
538
-
2290 2293 2295
2105
645 -646
647 -649
HY .4TN
2109 2112
+A +C +A
2775
A -',T
867
870
D -V
3086 -8
+ AT1T
970
974
+I
2957 3265
+TG +T
Tlhe single letter amino acid code is used.
SIAGAKKPFI
RNVFLN
Nucleotide sequence of the POL3 gene encoding DNA polymerase III (6) of Saccharomyces cerevisiae Alan Morrison and Akio Sugino Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, PO Box 12233, Research Triangle Park, NC 27709, USA Submitted September 16, 1991 We have described mutational studies of the conserved 3'- 5' exonuclease domain of S. cerevisiae DNA polymerase II (e) (1). On extending these studies to S. cerevisiae DNA polymerase III (6) in experiments similar to those now reported by Simon et al. (2), we noticed several differences between the nucleotide sequence of part of the POL3 gene (formerly CDC2) determined by us and the original sequence of Boulet et al. (3). Some of these differences were outside of the BspEI-BspEI fragment (nucleotides 973-1350) used for site-directed mutagenesis, and therefore had not arisen during this procedure. Neither was polymorphism the likely explanation, since we were using the POL3 plasmid pBL304, constructed in P.M.J.Burgers' laboratory and derived from pAB15 described by Boulet et al. (3). We considered whether these differences might have been mutations that had appeared during propagation of plasmid DNA in E. coli since the POL3 gene renders plasmids unstable. One of the nucleotide sequence differences, G923-A, removed a ThaI cleavage sequence present in the original sequence. We observed that ThaI did not cut at this site either in pBL304 or in yeast chromosomal DNA amplified by the polymerase chain reaction. Subsequently, we determined the nucleotide sequence of the entire POL3 gene of pBL304 by sequencing double stranded DNA using synthetic oligonucleotide primers and Sequenase (United States Biochemical Corporation). In addition we sequenced an independently isolated POL3 clone, HL1, provided by D.A.Gordenin. HL1 was isolated from a plasmid library of yeast genomic DNA by complementation of the tex] allele of POL3 (4). These two POL3 nucleotide sequences were identical. Table I summarizes the differences between the original and new sequences. The new nucleotide sequence begins at the MluI site and extends for 3551 nucleotides, corresponding to positions 193-3730 of the original sequence. The DNA polymerase III polypeptide predicted by the new sequence is 1097 amino acids long and differs from that described in reference 3 in twenty-six amino acid positions. None of the changes, however, occurs within the conserved motifs of the 3'-5' exonuclease or
EMBL accession no. X61920 polymerase domains. The biggest difference is in a sequence of seventeen residues (347 -363) occurring between the 'ExoI' and 'ExoHI motifs (1, 2).
REFERENCES 1. Morrison,A., Bell,J.B., Kunkel,T.A. and Sugino,A. (1991) Proc. Natl. Acad. Sci. USA 88, 9473-9477. 2. Simon,M., Giot,L. and Faye,G. (1991) EMBO J. 10, 2165-2170. 3. Boulet,A., Simon,M., Faye,G., Bauer,G.A. and Burgers,P.M.J. (1989) EMBO J. 8, 1849-1854. 4. Gordenin,D.A., Malkova,A.L., Peterzen,A., Kulikov,V.N., Pavlov,Y.I., Perkins,E. and Resnick,M.A. (1991) Proc. Natl. Acad. Sci. USA 88 (in press).
Table 1. Changes to the PKX3 sequence. I
S uoe ---de New positton Charge
Amino Acid Se_afe New positn Change 0
339
147
+T
Old position -
-
-
591
400
C -G
78
78
D-E
Old positon
592
401
G -C
79
79
V-+L
923
732
G -A
189
189
R-+H
968
777
A-+G
204
204
D -G
1059
868
A-T
234
234
-
1396 1399 1433 1440
1205 -6
+ AG
347 - 361
347 - 363
1210-11 1246 1254
YLALRNHSF VMCYSD -.
+A
1965
1780
T-+A
536
538
-
2290 2293 2295
2105
645 -646
647 -649
HY .4TN
2109 2112
+A +C +A
2775
A -',T
867
870
D -V
3086 -8
+ AT1T
970
974
+I
2957 3265
+TG +T
Tlhe single letter amino acid code is used.
SIAGAKKPFI
RNVFLN
