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Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions

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Journal of The American Society for Mass Spectrometry

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.

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Acknowledgments

C.E.M. was the recipient of an Australian Postgraduate Scholarship. This work was supported by grants from the Australian Research Council, including an Australian Professorial Fellowship to N.E.D.

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  1. School of Chemistry, University of Wollongong, Wollongong, NSW, Australia

    Claire E. Mason, Slobodan Jergic, Allen T. Y. Lo, Yao Wang, Nicholas E. Dixon & Jennifer L. Beck

  2. Research School of Chemistry, Australian National University, Canberra, ACT, Australia

    Nicholas E. Dixon

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  1. Claire E. Mason
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  2. Slobodan Jergic
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Correspondence to Jennifer L. Beck.

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Mason, C.E., Jergic, S., Lo, A.T.Y. et al. Escherichia coli Single-Stranded DNA-Binding Protein: NanoESI-MS Studies of Salt-Modulated Subunit Exchange and DNA Binding Transactions. J. Am. Soc. Mass Spectrom. 24, 274–285 (2013). https://doi.org/10.1007/s13361-012-0552-2

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