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

Ghosh, Shreya; Goldgur, Yehuda; Shuman, Stewart

doi:10.1093/nar/gkaa075

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

Journal Article · Sat Feb 08 00:00:00 EST 2020 · Nucleic Acids Research

DOI:https://doi.org/10.1093/nar/gkaa075· OSTI ID:1599461

Ghosh, Shreya ^[1]; Goldgur, Yehuda ^[2]; ^[1]

Molecular Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA
Structural Biology Program, Sloan-Kettering Institute, New York, NY 10065, USA

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.

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Research Organization:: Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)

Sponsoring Organization:: National Cancer Institute (NCI); National Institutes of Health (NIH); USDOE

Grant/Contract Number:: AC02-06CH11357

OSTI ID:: 1599461

Alternate ID(s):: OSTI ID: 1618479

Journal Information:: Nucleic Acids Research, Journal Name: Nucleic Acids Research Journal Issue: 6 Vol. 48; ISSN 0305-1048

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

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