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Crystal structure of DNA polymerase I from Thermus phage G20c

Ahlqvist, Josefin LU ; Linares-Pastén, Javier A. LU orcid ; Jasilionis, Andrius LU ; Welin, Martin LU ; Håkansson, Maria LU ; Svensson, L. Anders ; Wang, Lei LU ; Watzlawick, Hildegard ; Ævarsson, Arnþór and Friðjónsson, Ólafur H. , et al. (2022) In Acta crystallographica. Section D, Structural biology 78(Pt 11). p.1384-1398
Abstract

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I... (More)

This study describes the structure of DNA polymerase I from Thermus phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed SβαR, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The SβαR motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to Thermus phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel SβαR motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from Geobacillus stearothermophilus (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from Thermus aquaticus.

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type
Contribution to journal
publication status
published
subject
keywords
DNA Polymerase I/chemistry, Phosphodiesterase I, Thermus, Taq Polymerase/chemistry, Bacteriophages, Escherichia coli
in
Acta crystallographica. Section D, Structural biology
volume
78
issue
Pt 11
pages
15 pages
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:36322421
  • scopus:85141889241
ISSN
2059-7983
DOI
10.1107/S2059798322009895
language
English
LU publication?
yes
additional info
open access.
id
fe7bcd3e-1374-4809-bb22-b60dc42e121f
date added to LUP
2022-11-14 11:39:12
date last changed
2024-06-13 20:36:28
@article{fe7bcd3e-1374-4809-bb22-b60dc42e121f,
  abstract     = {{<p>This study describes the structure of DNA polymerase I from <i>Thermus</i> phage G20c, termed PolI_G20c. This is the first structure of a DNA polymerase originating from a group of related thermophilic bacteriophages infecting Thermus thermophilus, including phages G20c, TSP4, P74-26, P23-45 and phiFA and the novel phage Tth15-6. Sequence and structural analysis of PolI_G20c revealed a 3'-5' exonuclease domain and a DNA polymerase domain, and activity screening confirmed that both domains were functional. No functional 5'-3' exonuclease domain was present. Structural analysis also revealed a novel specific structure motif, here termed S<i>βα</i>R, that was not previously identified in any polymerase belonging to the DNA polymerases I (or the DNA polymerase A family). The S<i>βα</i>R motif did not show any homology to the sequences or structures of known DNA polymerases. The exception was the sequence conservation of the residues in this motif in putative DNA polymerases encoded in the genomes of a group of thermophilic phages related to <i>Thermus </i>phage G20c. The structure of PolI_G20c was determined with the aid of another structure that was determined in parallel and was used as a model for molecular replacement. This other structure was of a 3'-5' exonuclease termed ExnV1. The cloned and expressed gene encoding ExnV1 was isolated from a thermophilic virus metagenome that was collected from several hot springs in Iceland. The structure of ExnV1, which contains the novel S<i>βα</i>R motif, was first determined to 2.19 Å resolution. With these data at hand, the structure of PolI_G20c was determined to 2.97 Å resolution. The structures of PolI_G20c and ExnV1 are most similar to those of the Klenow fragment of DNA polymerase I (PDB entry 2kzz) from Escherichia coli, DNA polymerase I from <i>Geobacillus stearothermophilus</i> (PDB entry 1knc) and Taq polymerase (PDB entry 1bgx) from <i>Thermus aquaticus</i>.</p>}},
  author       = {{Ahlqvist, Josefin and Linares-Pastén, Javier A. and Jasilionis, Andrius and Welin, Martin and Håkansson, Maria and Svensson, L. Anders and Wang, Lei and Watzlawick, Hildegard and Ævarsson, Arnþór and Friðjónsson, Ólafur H. and Hreggviðsson, Guðmundur Ó. and Ketelsen Striberny, Bernd and Glomsaker, Eirin and Lanes, Olav and Al-Karadaghi, Salam and Nordberg Karlsson, Eva}},
  issn         = {{2059-7983}},
  keywords     = {{DNA Polymerase I/chemistry; Phosphodiesterase I; Thermus; Taq Polymerase/chemistry; Bacteriophages; Escherichia coli}},
  language     = {{eng}},
  month        = {{11}},
  number       = {{Pt 11}},
  pages        = {{1384--1398}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Acta crystallographica. Section D, Structural biology}},
  title        = {{Crystal structure of DNA polymerase I from <i>Thermus </i>phage G20c}},
  url          = {{http://dx.doi.org/10.1107/S2059798322009895}},
  doi          = {{10.1107/S2059798322009895}},
  volume       = {{78}},
  year         = {{2022}},
}