scholarly journals 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

2020 ◽  
Vol 48 (6) ◽  
pp. 3165-3180
Author(s):  
Shreya Ghosh ◽  
Yehuda Goldgur ◽  
Stewart Shuman
Keyword(s):  
Crystal Structures ◽  
Dna Polymerase ◽  
Ternary Complex ◽  
Structural Features ◽  
Reverse Transcriptase Activity ◽  
Dna Polymerase I ◽  
Polymerase Domain ◽  
Open Conformation ◽  
Rna:Dna Hybrid ◽  
Polymerase I

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.

scholarly journals Crystal structures of DNA polymerase I capture novel intermediates in the DNA synthesis pathway

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Nicholas Chim ◽  
Lynnette N Jackson ◽  
Anh M Trinh ◽  
John C Chaput
Keyword(s):  
High Resolution ◽  
Dna Synthesis ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Active Site ◽  
Dna Polymerase I ◽  
Dynamic Nature ◽  
Enzyme Active Site ◽  
Polymerase I ◽  
In Cells

High resolution crystal structures of DNA polymerase intermediates are needed to study the mechanism of DNA synthesis in cells. Here we report five crystal structures of DNA polymerase I that capture new conformations for the polymerase translocation and nucleotide pre-insertion steps in the DNA synthesis pathway. We suggest that these new structures, along with previously solved structures, highlight the dynamic nature of the finger subdomain in the enzyme active site.

Polymerase Domains of Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Herpes Simplex Virus Type 1 DNA Polymerase: Their Predicted Three-Dimensional Structures and some Putative Functions in Comparison with E. Coli DNA Polymerase I. A Critical Survey

1992 ◽  
Vol 3 (4) ◽  
pp. 223-241 ◽  
Author(s):  
B. Lindborg
Keyword(s):  
Dna Polymerase ◽  
Three Dimensional ◽  
Dna Polymerase I ◽  
E Coli ◽  
Polymerase Domain ◽  
Pol I ◽  
Hiv Rt ◽  
Polymerase I ◽  
Β Sheet

Hypothetical three-dimensional models for the entire polymerase domain of HIV-1 reverse transcriptase (HIV RT) and conserved regions of HSV-1 DNA polymerase (HSV pol) were created, primarily from literature data on mutations and principles of protein structure, and compared with those of E. coli DNA polymerase I (E. coli pol I). The corresponding parts, performing similar functions, were found to be analogous, not homologous, in structure with different β topologies and sequential arrangement. The polymerase domain of HSV pol is shown to form an anti-parallel β-sheet with α-helices, but with a topology different from that of the Klenow fragment of E. coli pol I. The main part of the polymerase domain of HIV RT is made up of a basically parallel β-sheet and α-helices with a topology similar to the nucleotide-binding p21 ras proteins. The putative functions of some conserved or invariant amino acids in the three polymerase families are discussed.

scholarly journals Crystal structures of a natural DNA polymerase that functions as an XNA reverse transcriptase

2019 ◽  
Vol 47 (13) ◽  
pp. 6973-6983 ◽  
Author(s):  
Lynnette N Jackson ◽  
Nicholas Chim ◽  
Changhua Shi ◽  
John C Chaput
Keyword(s):  
Nucleic Acid ◽  
Dna Synthesis ◽  
Crystal Structures ◽  
Dna Polymerase ◽  
Nucleoside Triphosphate ◽  
Dna Polymerase I ◽  
Bacterial Dna ◽  
Enzyme Active Site ◽  
Polymerase I ◽  
Innate Ability

Abstract Replicative DNA polymerases are highly efficient enzymes that maintain stringent geometric control over shape and orientation of the template and incoming nucleoside triphosphate. In a surprising twist to this paradigm, a naturally occurring bacterial DNA polymerase I member isolated from Geobacillus stearothermophilus (Bst) exhibits an innate ability to reverse transcribe RNA and other synthetic congeners (XNAs) into DNA. This observation raises the interesting question of how a replicative DNA polymerase is able to recognize templates of diverse chemical composition. Here, we present crystal structures of natural Bst DNA polymerase that capture the post-translocated product of DNA synthesis on templates composed entirely of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) and α-l-threofuranosyl nucleic acid (TNA). Analysis of the enzyme active site reveals the importance of structural plasticity as a possible mechanism for XNA-dependent DNA synthesis and provides insights into the construction of variants with improved activity.

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