Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA
| Autor: | Igor Konieczny, Urszula Walkow, Rafal Dutkiewicz, Jacek Czub, Miłosz Wieczór, María Moreno-del Álamo, Bartlomiej Tomiczek, Janusz M. Bujnicki, Igor Grochowina, Katarzyna Bury, Katarzyna Wegrzyn, Elzbieta Zabrocka, Rafael Giraldo, Urszula Uciechowska |
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| Přispěvatelé: | National Science Centre (Poland), Foundation for Polish Science, International Institute of Molecular and Cell Biology (Poland), Ministerio de Economía, Industria y Competitividad (España), Wegrzyn, Katarzyna, Tomiczek, Bartlomiej, Wieczor, Milosz, Czub, Jacek, Moreno-del Álamo, María, Grochowina, Igor, Dutkiewicz, Rafal, Giraldo, R., Konieczny, Igor, Wegrzyn, Katarzyna [0000-0002-3743-8597], Tomiczek, Bartlomiej [0000-0001-9295-663X], Wieczor, Milosz [0000-0003-4990-8629], Czub, Jacek [0000-0003-3639-6935], Moreno-del Álamo, María [0000-0002-6780-9686], Grochowina, Igor [0000-0003-2825-5315], Dutkiewicz, Rafal [0000-0003-4150-0450], Giraldo, R. [0000-0002-5358-7488], Konieczny, Igor [0000-0002-1588-5601] |
| Rok vydání: | 2021 |
| Předmět: |
Models
Molecular Protein family AcademicSubjects/SCI00010 Mutant DNA Helicases Sequence (biology) DNA Winged Helix Biology In vitro Cell biology DNA-Binding Proteins chemistry.chemical_compound Plasmid chemistry Protein Domains Replication Initiation Genetics Trans-Activators Molecular Biology Protein Binding |
| Zdroj: | Nucleic Acids Research Digital.CSIC. Repositorio Institucional del CSIC instname |
| ISSN: | 1362-4962 |
| Popis: | 15 p.-6 fig. An essential feature of replication initiation proteins is their ability to bind to DNA. In this work, we describe a new domain that contributes to a replication initiator sequence-specific interaction with DNA. Applying biochemical assays and structure prediction methods coupled with DNA-protein crosslinking, mass spectrometry, and construction and analysis of mutant proteins, we identified that the replication initiator of the broad host range plasmid RK2, in addition to two winged helix domains, contains a third DNA-binding domain. The phylogenetic analysis revealed that the composition of this unique domain is typical within the described TrfA-like protein family. Both in vitro and in vivo experiments involving the constructed TrfA mutant proteins showed that the newly identified domain is essential for the formation of the protein complex with DNA, contributes to the avidity for interaction with DNA, and the replication activity of the initiator. The analysis of mutant proteins, each containing a single substitution, showed that each of the three domains composing TrfA is essential for the formation of the protein complex with DNA. Furthermore, the new domain, along with the winged helix domains, contributes to the sequence specificity of replication initiator interaction within the plasmid replication origin. National Science Centre [2012/04/A/NZ1/00048 to I.K.;2017/26/D/NZ1/00239 to K.W.]; Foundation for Polish Science [TEAM, POIR.04.04.00-00-5C75/17-00 to I.K.]; International Institute of Molecular and Cell Biology in Warsaw (to J.M.B.); Ministerio de Economía,Industria y Competitividad (MINECO/AEI) [BIO2012-30852, RTI2018-094549-B-I00 to R.G.]. Funding for open access charge: Foundation for Polish Science [TEAM,POIR.04.04.00-00-5C75/17-00]. |
| Databáze: | OpenAIRE |
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