A sensor complements the steric gate when DNA polymerase ϵ discriminates ribonucleotides
Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › fagfællebedømt
Dokumenter
- Fulltext
Forlagets udgivne version, 1,99 MB, PDF-dokument
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.
| Originalsprog | Engelsk |
|---|---|
| Tidsskrift | Nucleic Acids Research |
| Vol/bind | 51 |
| Udgave nummer | 20 |
| Sider (fra-til) | 11225-11238 |
| ISSN | 0305-1048 |
| DOI | |
| Status | Udgivet - 2023 |
Bibliografisk note
Funding Information:
Knut and Alice Wallenberg Foundation [2018.0140, 2019.0431 to S.C.L.K.]; Swedish Research Council [2019-03499 to S.C.L.K., 2021-01104 to E.J.]; Swedish Cancer Society (to E.J.); the computations were enabled by resources provided by the Swedish National Infrastructure for Computing (SNIC), which is partially funded by the Swedish Research Council [2018-05973]. Funding for open access charge: Swedish universities (BIBSAM affiliated) (Sweden) agreement with OUP.
Funding Information:
We thank Alexandre Barrozo for help with establishing the system for Pol2 in the molecular dynamics simulations. Experiments were performed on beamlines ID29 and ID30A-3 at the European Synchrotron Radiation facility (ESRF), Grenoble. We are grateful to the local contact at ESRF for providing assistance in using beamlines ID29 and ID30A-3. We also acknowledge the MAX IV Laboratory for time on the BioMAX beamline under Proposal 20180236. Research conducted at MAX IV, a Swedish national user facility, is supported by the Swedish Research council under contract 2018-07152, the Swedish Governmental Agency for Innovation Systems under contract 2018-04969, and Formas under contract 2019-02496. Finally, we acknowledge DESY (Hamburg, Germany), a member of the Helmholtz Association HGF, for the provision of experimental facilities. Parts of this research were carried out at P13-PETRA III, and we would like to thank Drs Johanna Hakanpää and Saravanan Panneerselvam for assistance in using the P13 beamline. Beamtime was allocated for proposal 2019-MX-696.
Publisher Copyright:
© 2023 The Author(s). Published by Oxford University Press on behalf of Nucleic Acids Research.
ID: 375589495