TY - JOUR
T1 - Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-Template stabilization at the polymerization active site
AU - Juarez-Quintero, Víctor
AU - Peralta-Castro, Antolín
AU - Benítez Cardoza, Claudia G.
AU - Ellenberger, Tom
AU - Brieba, Luis G.
N1 - Publisher Copyright:
© 2021 Portland Press Ltd. All rights reserved.
PY - 2021/7
Y1 - 2021/7
N2 - 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 ?-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 ?-hairpin is involved in nucleotide incorporation on substrates with 50-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 30 50 exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating ?-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.
AB - 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 ?-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 ?-hairpin is involved in nucleotide incorporation on substrates with 50-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 30 50 exonuclease domain fold between family-A DNA polymerases and highlight the modular architecture of T7 DNA polymerase. Our data suggest that the intercalating ?-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.
UR - http://www.scopus.com/inward/record.url?scp=85110652131&partnerID=8YFLogxK
U2 - 10.1042/BCJ20200922
DO - 10.1042/BCJ20200922
M3 - Artículo
C2 - 34160020
AN - SCOPUS:85110652131
SN - 0264-6021
VL - 478
SP - 2665
EP - 2679
JO - Biochemical Journal
JF - Biochemical Journal
IS - 13
ER -