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Primer‐terminus stabilization at the 3′‐5′ exonuclease active site of phi29 DNA polymerase. Involvement of two amino acid residues highly conserved in proofreading DNA polymerases.
Author(s) -
Vega M.,
Lazaro J. M.,
Salas M.,
Blanco L.
Publication year - 1996
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/j.1460-2075.1996.tb00457.x
Subject(s) - dna polymerase , biology , primer (cosmetics) , dna polymerase ii , primase , klenow fragment , dna clamp , polymerase , exonuclease , dna polymerase i , biochemistry , proofreading , dna , dna polymerase mu , dna replication , microbiology and biotechnology , circular bacterial chromosome , chemistry , reverse transcriptase , rna , gene , organic chemistry
By site‐directed mutagenesis in phi29 DNA polymerase, we have analyzed the functional importance of two evolutionarily conserved residues belonging to the 3′‐5′ exonuclease domain of DNA‐dependent DNA polymerases. In Escherichia coli DNA polymerase I, these residues are Thr358 and Asn420, shown by crystallographic analysis to be directly acting as single‐stranded DNA (ssDNA) ligands at the 3′‐5′ exonuclease active site. On the basis of these structural data, single substitution of the corresponding residues of phi29 DNA polymerase, Thr15 and Asn62, produced enzymes with a very reduced or altered capacity to bind ssDNA. Analysis of the residual 3′‐5′ exonuclease activity of these mutant derivatives on ssDNA substrates allowed us to conclude that these two residues do not play a direct role in the catalysis of the reaction. On the other hand, analysis of the 3′‐5′ exonuclease activity on either matched or mismatched primer/template structures showed a critical role of these two highly conserved residues in exonucleolysis under polymerization conditions, i.e. in the proofreading of DNA polymerization errors, an evolutionary advantage of most DNA‐dependent DNA polymerases. Moreover, in contrast to the dual role in 3′‐5′ exonucleolysis and strand displacement previously observed for phi29 DNA polymerase residues acting as metal ligands, the contribution of residues Thr15 and Asn62 appears to be restricted to the proofreading function, by stabilization of the frayed primer‐terminus at the 3′‐5′ exonuclease active site.