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.
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Cover Image
Cover Image
SMCHD1 is an epigenetic regulator that mediates gene expression silencing at targeted sites across the genome. In this issue Gurzau and colleagues (pp. 2555–2569) use biophysical techniques to we demonstrate that the SMCHD1 ATPase undergoes dimerization in a process that is dependent on both the N-terminal Ubiquitin-like domain and ATP binding. The cover image shows immunofluorescence with DAPI staining in cyan and SMCHD1 showing in yellow. Scale bars are 20 µm. Image provided by James M. Murphy.
Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site
Víctor Juarez-Quintero, Antolín Peralta-Castro, Claudia G. Benítez Cardoza, Tom Ellenberger, Luis G. Brieba; Structure of an open conformation of T7 DNA polymerase reveals novel structural features regulating primer-template stabilization at the polymerization active site. Biochem J 16 July 2021; 478 (13): 2665–2679. doi: https://doi.org/10.1042/BCJ20200922
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