title: Novel, fluorescent, SSB protein chimeras with broad utility.
abstract: The Escherichia coli single-stranded DNA binding protein (SSB) is a central player in DNA metabolism where it organizes genome maintenance complexes and stabilizes single-stranded DNA (ssDNA) intermediates generated during DNA processing. Due to the importance of SSB and to facilitate real-time studies, we developed a dual plasmid expression system to produce novel, chimeric SSB proteins. These chimeras, which contain mixtures of histidine-tagged and fluorescent protein(FP)-fusion subunits, are easily purified in milligram quantities and used without further modification, a significant enhancement over previous methods to produce fluorescent SSB. Chimeras retain the functionality of wild type in all assays, demonstrating that SSB function is unaffected by the FPs. We demonstrate the power and utility of these chimeras in single molecule studies providing a great level of insight into the biochemical mechanism of RecBCD. We also utilized the chimeras to show for the first time that RecG and SSB interact in vivo. Consequently, we anticipate that the chimeras described herein will facilitate in vivo, in vitro and single DNA molecule studies using proteins that do not require further modification prior to use.
authors.affiliation: Center for Single Molecule Biophysics, Department of Microbiology and Immunology, University at Buffalo, Buffalo, New York 14214, USA.
authors.forename: Juan
authors.initials: J
authors.lastname: Liu
authors.forename: Meerim
authors.initials: M
authors.lastname: Choi
authors.forename: Adam G
authors.initials: AG
authors.lastname: Stanenas
authors.forename: Alicia K
authors.initials: AK
authors.lastname: Byrd
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Raney
authors.forename: Christopher
authors.initials: C
authors.lastname: Cohan
authors.forename: Piero R
authors.initials: PR
authors.lastname: Bianco
mesh_terms: D003001:Cloning, Molecular; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins