EUKARYOTIC DNA-POLYMERASE AMINO-ACID-SEQUENCE REQUIRED FOR 3'-] 5' EXONUCLEASE ACTIVITY

被引:263
作者
MORRISON, A
BELL, JB
KUNKEL, TA
SUGINO, A
机构
[1] Molecular Genetics Laboratory, Natl. Envtl. Health Sci. Inst., Res. Triangle Park, NC 27709
关键词
YEAST DNA POLYMERASE-II; MUTATOR PHENOTYPE; PROOFREADING; PROTEIN DOMAIN; DNA REPLICATION;
D O I
10.1073/pnas.88.21.9473
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
We have identified an amino-proximal sequence motif, Phe-Asp-Ile-Glu-Thr, in Saccharomyces cerevisiae DNA polymerase II that is almost identical to a sequence comprising part of the 3' --> 5' exonuclease active site of Escherichia coli DNA polymerase I. Similar motifs were identified by amino acid sequence alignment in related, aphidicolin-sensitive DNA polymerases possessing 3' --> 5' proofreading exonuclease activity. Substitution of Ala for the Asp and Glu residues in the motif reduced the exonuclease activity of partially purified DNA polymerase II at least 100-fold while preserving the polymerase activity. Yeast strains expressing the exonuclease-deficient DNA polymerase II had on average about a 22-fold increase in spontaneous mutation rate, consistent with a presumed proofreading role in vivo. In multiple amino acid sequence alignments of this and two other conserved motifs described previously, five residues of the 3' --> 5' exonuclease active site of E. coli DNA polymerase I appeared to be invariant in aphidicolin-sensitive DNA polymerases known to possess 3' --> 5' proofreading exonuclease activity. None of these residues, however, appeared to be identifiable in the catalytic subunits of human, yeast, or Drosophila alpha-DNA polymerases.
引用
收藏
页码:9473 / 9477
页数:5
相关论文
共 33 条
[1]   A CONSERVED 3'-]5' EXONUCLEASE ACTIVE-SITE IN PROKARYOTIC AND EUKARYOTIC DNA-POLYMERASES [J].
BERNAD, A ;
BLANCO, L ;
LAZARO, JM ;
MARTIN, G ;
SALAS, M .
CELL, 1989, 59 (01) :219-228
[2]   THE HIGHLY CONSERVED AMINO-ACID-SEQUENCE MOTIF TYR-GLY-ASP-THR-ASP-SER IN ALPHA-LIKE DNA-POLYMERASES IS REQUIRED BY PHAGE-PHI-29 DNA-POLYMERASE FOR PROTEIN-PRIMED INITIATION AND POLYMERIZATION [J].
BERNAD, A ;
LAZARO, JM ;
SALAS, M ;
BLANCO, L .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1990, 87 (12) :4610-4614
[3]   A POSITIVE SELECTION FOR MUTANTS LACKING OROTIDINE-5'-PHOSPHATE DECARBOXYLASE ACTIVITY IN YEAST - 5-FLUORO-OROTIC ACID RESISTANCE [J].
BOEKE, JD ;
LACROUTE, F ;
FINK, GR .
MOLECULAR & GENERAL GENETICS, 1984, 197 (02) :345-346
[4]   STRUCTURE AND FUNCTION OF THE SACCHAROMYCES-CEREVISIAE CDC2 GENE ENCODING THE LARGE SUBUNIT OF DNA POLYMERASE-III [J].
BOULET, A ;
SIMON, M ;
FAYE, G ;
BAUER, GA ;
BURGERS, PMJ .
EMBO JOURNAL, 1989, 8 (06) :1849-1854
[5]  
BROOKE RG, 1991, J BIOL CHEM, V266, P3005
[6]  
BUDD ME, 1989, J BIOL CHEM, V264, P6557
[7]  
CHANG LMS, 1977, J BIOL CHEM, V252, P1873
[8]   AN ATTEMPT TO UNIFY THE STRUCTURE OF POLYMERASES [J].
DELARUE, M ;
POCH, O ;
TORDO, N ;
MORAS, D ;
ARGOS, P .
PROTEIN ENGINEERING, 1990, 3 (06) :461-467
[9]   THE 3'-5' EXONUCLEASE OF DNA-POLYMERASE-I OF ESCHERICHIA-COLI - CONTRIBUTION OF EACH AMINO-ACID AT THE ACTIVE-SITE TO THE REACTION [J].
DERBYSHIRE, V ;
GRINDLEY, NDF ;
JOYCE, CM .
EMBO JOURNAL, 1991, 10 (01) :17-24
[10]   GENETIC AND CRYSTALLOGRAPHIC STUDIES OF THE 3',5'-EXONUCLEOLYTIC SITE OF DNA-POLYMERASE-I [J].
DERBYSHIRE, V ;
FREEMONT, PS ;
SANDERSON, MR ;
BEESE, L ;
FRIEDMAN, JM ;
JOYCE, CM ;
STEITZ, TA .
SCIENCE, 1988, 240 (4849) :199-201