Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme
Abstract
Abstract Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.
- Authors:
-
[1]
- Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
- Structural Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
- Publication Date:
- Sponsoring Org.:
- USDOE
- OSTI Identifier:
- 1599461
- Grant/Contract Number:
- AC02-06CH11357
- Resource Type:
- Published Article
- Journal Name:
- Nucleic Acids Research
- Additional Journal Information:
- Journal Name: Nucleic Acids Research; Journal ID: ISSN 0305-1048
- Publisher:
- Oxford University Press
- Country of Publication:
- United Kingdom
- Language:
- English
Citation Formats
Ghosh, Shreya, Goldgur, Yehuda, and Shuman, Stewart. Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme. United Kingdom: N. p., 2020.
Web. doi:10.1093/nar/gkaa075.
Ghosh, Shreya, Goldgur, Yehuda, & Shuman, Stewart. Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme. United Kingdom. doi:10.1093/nar/gkaa075.
Ghosh, Shreya, Goldgur, Yehuda, and Shuman, Stewart. Sat .
"Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme". United Kingdom. doi:10.1093/nar/gkaa075.
@article{osti_1599461,
title = {Mycobacterial DNA polymerase I: activities and crystal structures of the POL domain as apoenzyme and in complex with a DNA primer-template and of the full-length FEN/EXO–POL enzyme},
author = {Ghosh, Shreya and Goldgur, Yehuda and Shuman, Stewart},
abstractNote = {Abstract Mycobacterial Pol1 is a bifunctional enzyme composed of an N-terminal DNA flap endonuclease/5′ exonuclease domain (FEN/EXO) and a C-terminal DNA polymerase domain (POL). Here we document additional functions of Pol1: FEN activity on the flap RNA strand of an RNA:DNA hybrid and reverse transcriptase activity on a DNA-primed RNA template. We report crystal structures of the POL domain, as apoenzyme and as ternary complex with 3′-dideoxy-terminated DNA primer-template and dNTP. The thumb, palm, and fingers subdomains of POL form an extensive interface with the primer-template and the triphosphate of the incoming dNTP. Progression from an open conformation of the apoenzyme to a nearly closed conformation of the ternary complex entails a disordered-to-ordered transition of several segments of the thumb and fingers modules and an inward motion of the fingers subdomain—especially the O helix—to engage the primer-template and dNTP triphosphate. Distinctive structural features of mycobacterial Pol1 POL include a manganese binding site in the vestigial 3′ exonuclease subdomain and a non-catalytic water-bridged magnesium complex at the protein-DNA interface. We report a crystal structure of the bifunctional FEN/EXO–POL apoenzyme that reveals the positions of two active site metals in the FEN/EXO domain.},
doi = {10.1093/nar/gkaa075},
journal = {Nucleic Acids Research},
number = ,
volume = ,
place = {United Kingdom},
year = {2020},
month = {2}
}
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DOI: 10.1093/nar/gkaa075
DOI: 10.1093/nar/gkaa075
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