A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides
Parkash, Vimal ; Kulkarni, Yashraj ; Bylund, Göran O. ; Osterman, Pia ; Kamerlin, Shina Caroline Lynn LU
and Johansson, Erik
(2023)
In Nucleic Acids Research
51(20).
p.11225-11238
- Abstract
The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the... (More)
The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.
(Less)
- author
- Parkash, Vimal
; Kulkarni, Yashraj
; Bylund, Göran O.
; Osterman, Pia
; Kamerlin, Shina Caroline Lynn
LU
and Johansson, Erik
- publishing date
- 2023-11-10
- type
- Contribution to journal
- publication status
- published
- keywords
- Catalytic Domain, Crystallography, X-Ray, Deoxyribonucleotides/metabolism, DNA/genetics, DNA Polymerase II/chemistry, Ribonucleotides/metabolism, Saccharomyces cerevisiae/enzymology
- in
- Nucleic Acids Research
- volume
- 51
- issue
- 20
- pages
- 14 pages
- publisher
- Oxford University Press
- external identifiers
-
- pmid:37819038
- scopus:85178042069
- ISSN
- 1362-4962
- DOI
- 10.1093/nar/gkad817
- language
- English
- LU publication?
- no
- additional info
- © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.
- id
- 557acd55-340b-435d-bbc5-fc5e8514ae0f
- date added to LUP
- 2025-01-11 18:39:09
- date last changed
- 2025-07-13 18:38:41
@article{557acd55-340b-435d-bbc5-fc5e8514ae0f,
abstract = {{<p>The cellular imbalance between high concentrations of ribonucleotides (NTPs) and low concentrations of deoxyribonucleotides (dNTPs), is challenging for DNA polymerases when building DNA from dNTPs. It is currently believed that DNA polymerases discriminate against NTPs through a steric gate model involving a clash between a tyrosine and the 2'-hydroxyl of the ribonucleotide in the polymerase active site in B-family DNA polymerases. With the help of crystal structures of a B-family polymerase with a UTP or CTP in the active site, molecular dynamics simulations, biochemical assays and yeast genetics, we have identified a mechanism by which the finger domain of the polymerase sense NTPs in the polymerase active site. In contrast to the previously proposed polar filter, our experiments suggest that the amino acid residue in the finger domain senses ribonucleotides by steric hindrance. Furthermore, our results demonstrate that the steric gate in the palm domain and the sensor in the finger domain are both important when discriminating NTPs. Structural comparisons reveal that the sensor residue is conserved among B-family polymerases and we hypothesize that a sensor in the finger domain should be considered in all types of DNA polymerases.</p>}},
author = {{Parkash, Vimal and Kulkarni, Yashraj and Bylund, Göran O. and Osterman, Pia and Kamerlin, Shina Caroline Lynn and Johansson, Erik}},
issn = {{1362-4962}},
keywords = {{Catalytic Domain; Crystallography, X-Ray; Deoxyribonucleotides/metabolism; DNA/genetics; DNA Polymerase II/chemistry; Ribonucleotides/metabolism; Saccharomyces cerevisiae/enzymology}},
language = {{eng}},
month = {{11}},
number = {{20}},
pages = {{11225--11238}},
publisher = {{Oxford University Press}},
series = {{Nucleic Acids Research}},
title = {{A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides}},
url = {{http://dx.doi.org/10.1093/nar/gkad817}},
doi = {{10.1093/nar/gkad817}},
volume = {{51}},
year = {{2023}},
}