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Interaction of a Dimeric Single-Stranded DNA-Binding Protein (G5P) with DNA Hairpins. A Molecular Beacon Study

  • Gene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein–DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction ofGene-V protein (G5P/GVP) is a single-stranded (ss)DNA-binding protein (SBP) of bacteriophage f1 that is required for DNA synthesis and repair. In solution, it exists as a dimer that binds two antiparallel ssDNA strands with high affinity in a cooperative manner, forming a left-handed helical protein–DNA filament. Here, we report on fluorescence studies of the interaction of G5P with different DNA oligonucleotides having a hairpin structure (molecular beacon, MB) with a seven base-pair stem (dT24-stem7, dT18-stem7), as well as with DNA oligonucleotides (dT38, dT24) without a defined secondary structure. All oligonucleotides were end-labeled with a Cy3-fluorophore and a BHQ2-quencher. In the case of DNA oligonucleotides without a secondary structure, an almost complete quenching of their strong fluorescence (with about 5% residual intensity) was observed upon the binding of G5P. This implies an exact alignment of the ends of the DNA strand(s) in the saturated complex. The interaction of the DNA hairpins with G5P led to the unzipping of the base-paired stem, as revealed by fluorescence measurements, fluorescence microfluidic mixing experiments, and electrophoretic mobility shift assay data. Importantly, the disruption of ssDNA’s secondary structure agrees with the behavior of other single-stranded DNA-binding proteins (SBPs). In addition, substantial protein-induced fluorescence enhancement (PIFE) of the Cy3-fluorescence was observed.zeige mehrzeige weniger

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Autor*innen:Tihomir Solomun, Leo Cordsmeier, Dorothea C. Hallier, Harald Seitz, Marc Benjamin HahnORCiD
Dokumenttyp:Zeitschriftenartikel
Veröffentlichungsform:Verlagsliteratur
Sprache:Englisch
Titel des übergeordneten Werkes (Englisch):The Journal of Physical Chemistry B
Jahr der Erstveröffentlichung:2023
Organisationseinheit der BAM:6 Materialchemie
6 Materialchemie / 6.6 Physik und chemische Analytik der Polymere
Verlag:ACS Publications
Jahrgang/Band:127
Ausgabe/Heft:38
Erste Seite:8131
Letzte Seite:8138
DDC-Klassifikation:Naturwissenschaften und Mathematik / Chemie / Analytische Chemie
Technik, Medizin, angewandte Wissenschaften / Ingenieurwissenschaften / Sanitär- und Kommunaltechnik; Umwelttechnik
Freie Schlagwörter:Amino Acids; Bacteriophage f1; Cy3; DNA; Fluorescence; G5P; Hairpin; Molecular beacon; Oligonucleotides; PIFE; Protein; Protein-induced fluorescence enhancement; Protein–DNA filament; Quencher; Raman; SBP; SSB; Single-stranded DNA-binding protein; ssDNA
Themenfelder/Aktivitätsfelder der BAM:Chemie und Prozesstechnik
Umwelt
Umwelt / Umwelt-Material-Interaktionen
DOI:10.1021/acs.jpcb.3c03669
ISSN:1520-6106
Verfügbarkeit des Dokuments:Datei im Netzwerk der BAM verfügbar ("Closed Access")
Datum der Freischaltung:18.09.2023
Referierte Publikation:Ja
Datum der Eintragung als referierte Publikation:04.10.2023
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