Open AccessStructure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site
Author(s) -
Víctor Juárez-Quintero,
Antolín Peralta-Castro,
Claudia Guadalupe Benítez Cardoza,
Tom Ellenberger,
Luis G. Brieba
Publication year - 2021
Publication title -
biochemical journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.706
H-Index - 265
eISSN - 1470-8728
pISSN - 0264-6021
DOI - 10.1042/bcj20200922
Subject(s) - primase , dna polymerase , dna clamp , dna polymerase ii , dna replication , primer (cosmetics) , coding strand , klenow fragment , biology , processivity , exonuclease , polymerase , dna , biochemistry , microbiology and biotechnology , biophysics , chemistry , rna , reverse transcriptase , organic chemistry , gene
The crystal structure of full-length T7 DNA polymerase in complex with its processivity factor thioredoxin and double-stranded DNA in the polymerization active site exhibits two novel structural motifs in family-A DNA polymerases: an extended β-hairpin at the fingers subdomain, that interacts with the DNA template strand downstream the primer-terminus, and a helix-loop-helix motif (insertion1) located between residues 102 to 122 in the exonuclease domain. The extended β-hairpin is involved in nucleotide incorporation on substrates with 5′-overhangs longer than 2 nt, suggesting a role in stabilizing the template strand into the polymerization domain. Our biochemical data reveal that insertion1 of the exonuclease domain makes stabilizing interactions that facilitate proofreading by shuttling the primer strand into the exonuclease active site. Overall, our studies evidence conservation of the 3′–5′ exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating β-hairpin guides the template-strand into the polymerization active site after the T7 primase-helicase unwinds the DNA double helix ameliorating the formation of secondary structures and decreasing the appearance of indels.