Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site

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 t...

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Veröffentlicht in:Biochemical journal 2021-07, Vol.478 (13), p.2665-2679
Hauptverfasser: Juarez-Quintero, Víctor, Peralta-Castro, Antolín, Benítez Cardoza, Claudia G., Ellenberger, Tom, Brieba, Luis G.
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container_end_page 2679
container_issue 13
container_start_page 2665
container_title Biochemical journal
container_volume 478
creator Juarez-Quintero, Víctor
Peralta-Castro, Antolín
Benítez Cardoza, Claudia G.
Ellenberger, Tom
Brieba, Luis G.
description 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.
doi_str_mv 10.1042/BCJ20200922
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title Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site
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