Point mutations V546E and D547H of the RBM-B motif does not affect the binding of PrimPol to RPA and DNA
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| 1. | Title | Title of document | Point mutations V546E and D547H of the RBM-B motif does not affect the binding of PrimPol to RPA and DNA |
| 2. | Creator | Author's name, affiliation, country | A. A. Manukyan; National Research Center “Kurchatov Institute”; Russian Federation |
| 2. | Creator | Author's name, affiliation, country | A. V. Makarova; National Research Center “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences; Russian Federation |
| 2. | Creator | Author's name, affiliation, country | E. O. Boldinova; National Research Center “Kurchatov Institute”; Institute of Gene Biology, Russian Academy of Sciences; Russian Federation |
| 3. | Subject | Discipline(s) | |
| 3. | Subject | Keyword(s) | primase-polymerase PrimPol; DNA synthesis restart; replication; RPA |
| 4. | Description | Abstract | The human primase-polymerase PrimPol is a key participant of the mechanism of DNA synthesis restart during replication fork stalling at DNA damaged sites. PrimPol has a DNA primase activity and synthesizes DNA primers that are used by processive DNA polymerases to continue replication. Recruitment of PrimPol to the sites of DNA damage, as well as catalytic activity stimulation depends on interaction with the replicative protein RPA, which binds single-stranded DNA. The C-terminal domain of PrimPol contains a negatively charged RPA-binding motif (RBM), which mutations disrupt the interaction between two proteins. The RBM motif also plays a role in the negative regulation of PrimPol interaction with DNA. Deletion of RBM dramatically increases PrimPol affinity to DNA and stimulates PrimPol activity. The mechanism of RBM-mediated regulation of PrimPol activity is unclear. The relatively strong negative charge of RBM potentially may contribute to the interaction of PrimPol with RPA and DNA. RBM contains two negatively charged regions RBM-A and RBM-B. In this work, we additionally added (substitution V546E) or decreased (substitution D547H) the negative charge in RBM-B PrimPol and characterized these mutant variants biochemically. It was shown that the local change of RBM-B charge has no effect on the interaction of PrimPol with DNA and RPA, as well as the catalytic activity of the enzyme. |
| 5. | Publisher | Organizing agency, location | The Russian Academy of Sciences |
| 6. | Contributor | Sponsor(s) |
Russian Science Foundation (22-24-20150) |
| 7. | Date | (DD-MM-YYYY) | 15.10.2024 |
| 8. | Type | Status & genre | Peer-reviewed Article |
| 8. | Type | Type | Research Article |
| 9. | Format | File format | |
| 10. | Identifier | Uniform Resource Identifier | https://rjraap.com/0026-8984/article/view/683307 |
| 10. | Identifier | Digital Object Identifier (DOI) | 10.31857/S0026898424050122 |
| 10. | Identifier | eLIBRARY Document Number (EDN) | HUAYQY |
| 11. | Source | Title; vol., no. (year) | Molekulârnaâ biologiâ; Vol 58, No 5 (2024) |
| 12. | Language | English=en | ru |
| 13. | Relation | Supp. Files |
Appendix (677KB) Fig. 1. Comparison of the catalytic activity of wild-type PrimPol and the V546E and D547H variants. a – Alignment of the amino acid sequences of RBM-A and RBM-B PrimPol from organisms of different taxa. Negatively charged Asp and Glu residues are marked in red. Orange stars indicate the PrimPol residues that form bonds with the RPA70 subunit, according to the structure [9]. б – DNA polymerase activity of PrimPol and variants with V546E and D547H substitutions. Reactions were carried out in the presence of 20/50/100/200/400/800 nM PrimPol for 10 min. в – DNA polymerase activity of PrimPol and its variants depending on the reaction time, as well as the kobs parameter value. Reactions were carried out in the presence of 200 nM PrimPol for 2/5/10/30/60/120 min. г – Total DNA primase activity of PrimPol and variants with V546E and D547H substitutions. Reactions were carried out in the presence of [γ-32P]ATP, ATP, dGTP and dTTP for 30/60/90 min. д – Formation of dinucleotide of wild-type PrimPol and variants V546E and D547H. Reactions were carried out in the presence of [γ-32P]ATP, ATP and dGTP for 30 min. (761KB) Fig. 2. Shift analysis of the PrimPol complex of wild type and variants with V546E and D547H substitutions with DNA in a native gel. Mean values and standard errors are shown. (317KB) Fig. 3. Analysis of the effect of V546E and D547H substitutions on the interaction of PrimPol with the replicative protein RPA. a – Formation of the ternary RPA:DNA:PrimPol complex in 5% native gel. б – Electropherogram of the primer extension reactions. DNA polymerase reactions were carried out in the presence of 100 nM PrimPol or its variants for 5/10/30 min. c – Graphs reflecting the efficiency of primer extension depending on the reaction time. Mean values and standard errors are shown. г – Coprecipitation of wild-type PrimPol and the V546E and D547H variants with the RPA protein. The red arrow indicates the position of the band of the RPA70 subunit that formed a complex with GST-PrimPol. (536KB) |
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| 15. | Rights | Copyright and permissions |
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