Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA.

Leila Shokri, Boriana Marintcheva, Mootaz Eldib, Andreas Hanke, Ioulia Rouzina, Mark C. Williams

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Bacteriophage T7 gene 2.5 protein (gp2.5) is a single-stranded DNA (ssDNA)-binding protein that has essential roles in DNA replication, recombination and repair. However, it differs from other ssDNA-binding proteins by its weaker binding to ssDNA and lack of cooperative ssDNA binding. By studying the rate-dependent DNA melting force in the presence of gp2.5 and its deletion mutant lacking 26 C-terminal residues, we probe the kinetics and thermodynamics of gp2.5 binding to ssDNA and double-stranded DNA (dsDNA). These force measurements allow us to determine the binding rate of both proteins to ssDNA, as well as their equilibrium association constants to dsDNA. The salt dependence of dsDNA binding parallels that of ssDNA binding. We attribute the four orders of magnitude salt-independent differences between ssDNA and dsDNA binding to nonelectrostatic interactions involved only in ssDNA binding, in contrast to T4 gene 32 protein, which achieves preferential ssDNA binding primarily through cooperative interactions. The results support a model in which dimerization interactions must be broken for DNA binding, and gp2.5 monomers search dsDNA by 1D diffusion to bind ssDNA. We also quantitatively compare the salt-dependent ssDNA- and dsDNA-binding properties of the T4 and T7 ssDNA-binding proteins for the first time.

Original languageEnglish (US)
Pages (from-to)5668-5677
Number of pages10
JournalNucleic acids research
Volume36
Issue number17
DOIs
StatePublished - Oct 2008

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    Shokri, L., Marintcheva, B., Eldib, M., Hanke, A., Rouzina, I., & Williams, M. C. (2008). Kinetics and thermodynamics of salt-dependent T7 gene 2.5 protein binding to single- and double-stranded DNA. Nucleic acids research, 36(17), 5668-5677. https://doi.org/10.1093/nar/gkn551