title: Escherichia coli single-stranded DNA-binding protein: nanoESI-MS studies of salt-modulated subunit exchange and DNA binding transactions.
abstract: Single-stranded DNA-binding proteins (SSBs) are ubiquitous oligomeric proteins that bind with very high affinity to single-stranded DNA and have a variety of essential roles in DNA metabolism. Nanoelectrospray ionization mass spectrometry (nanoESI-MS) was used to monitor subunit exchange in full-length and truncated forms of the homotetrameric SSB from Escherichia coli. Subunit exchange in the native protein was found to occur slowly over a period of hours, but was significantly more rapid in a truncated variant of SSB from which the eight C-terminal residues were deleted. This effect is proposed to result from C-terminus mediated stabilization of the SSB tetramer, in which the C-termini interact with the DNA-binding cores of adjacent subunits. NanoESI-MS was also used to examine DNA binding to the SSB tetramer. Binding of single-stranded oligonucleotides [one molecule of (dT)(70), one molecule of (dT)(35), or two molecules of (dT)(35)] was found to prevent SSB subunit exchange. Transfer of SSB tetramers between discrete oligonucleotides was also observed and is consistent with predictions from solution-phase studies, suggesting that SSB-DNA complexes can be reliably analyzed by ESI mass spectrometry.
authors.affiliation: School of Chemistry, University of Wollongong, Wollongong, NSW, Australia.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Mason
authors.forename: Slobodan
authors.initials: S
authors.lastname: Jergic
authors.forename: Allen T Y
authors.initials: AT
authors.lastname: Lo
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Dixon
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Beck
mesh_terms: D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D036103:Nanotechnology; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D019346:Sodium Acetate / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D009841:Oligonucleotides; D021122:Protein Subunits; D019346:Sodium Acetate