Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site
| Autor: | Tom Ellenberger, Claudia Guadalupe Benítez Cardoza, Luis G. Brieba, Antolín Peralta-Castro, Víctor Juarez-Quintero |
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| Rok vydání: | 2021 |
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Molecular Exonuclease Protein Conformation DNA polymerase DNA-Directed DNA Polymerase Biochemistry Protein Structure Secondary Polymerization Viral Proteins 03 medical and health sciences chemistry.chemical_compound Protein Domains Bacteriophage T7 Catalytic Domain Amino Acid Sequence Molecular Biology DNA Primers 030304 developmental biology 0303 health sciences Sequence Homology Amino Acid biology Chemistry 030302 biochemistry & molecular biology T7 DNA polymerase DNA Templates Genetic Cell Biology Processivity Coding strand Mutation biology.protein Biophysics Nucleic Acid Conformation Replisome Primer (molecular biology) Protein Binding |
| Zdroj: | Biochemical Journal. 478:2665-2679 |
| ISSN: | 1470-8728 0264-6021 |
| DOI: | 10.1042/bcj20200922 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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