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Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA
Abstract
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.
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Authors (14)
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Katarzyna Wegrzyn
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Elzbieta Zabrocka
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Katarzyna Bury
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Bartlomiej Tomiczek
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Urszula Uciechowska
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María Moreno-del Alamo
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Urszula Walkow
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Igor Grochowina
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Rafal Dutkiewicz
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Janusz M. Bujnicki
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Rafael Giraldo
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Igor Konieczny
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Details
- Category:
- Articles
- Type:
- artykuły w czasopismach
- Published in:
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NUCLEIC ACIDS RESEARCH
no. 49,
pages 3394 - 3408,
ISSN: 0305-1048 - Language:
- English
- Publication year:
- 2021
- Bibliographic description:
- Wegrzyn K., Zabrocka E., Bury K., Tomiczek B., Wieczór M., Czub J., Uciechowska U., Moreno-Del alamo M., Walkow U., Grochowina I., Dutkiewicz R., Bujnicki J., Giraldo R., Konieczny I.: Defining a novel domain that provides an essential contribution to site-specific interaction of Rep protein with DNA// NUCLEIC ACIDS RESEARCH -Vol. 49,iss. 6 (2021), s.3394-3408
- DOI:
- Digital Object Identifier (open in new tab) 10.1093/nar/gkab113
- Verified by:
- Gdańsk University of Technology
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