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Development of a single-stranded DNA-binding protein fluorescent fusion toolbox

Journal Article


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Abstract


  • © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Authors


Publication Date


  • 2020

Citation


  • Dubiel, K., Henry, C., Spenkelink, L., Kozlov, A., Wood, E., Jergic, S., Dixon, N., van Oijen, A., Cox, M., Lohman, T., Sandler, S. & Keck, J. (2020). Development of a single-stranded DNA-binding protein fluorescent fusion toolbox. Nucleic acids research, 48 (11), 6053-6067.

Scopus Eid


  • 2-s2.0-85086525296

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2439&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1405

Number Of Pages


  • 14

Start Page


  • 6053

End Page


  • 6067

Volume


  • 48

Issue


  • 11

Place Of Publication


  • United Kingdom

Abstract


  • © The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Authors


Publication Date


  • 2020

Citation


  • Dubiel, K., Henry, C., Spenkelink, L., Kozlov, A., Wood, E., Jergic, S., Dixon, N., van Oijen, A., Cox, M., Lohman, T., Sandler, S. & Keck, J. (2020). Development of a single-stranded DNA-binding protein fluorescent fusion toolbox. Nucleic acids research, 48 (11), 6053-6067.

Scopus Eid


  • 2-s2.0-85086525296

Ro Full-text Url


  • https://ro.uow.edu.au/cgi/viewcontent.cgi?article=2439&context=smhpapers1

Ro Metadata Url


  • http://ro.uow.edu.au/smhpapers1/1405

Number Of Pages


  • 14

Start Page


  • 6053

End Page


  • 6067

Volume


  • 48

Issue


  • 11

Place Of Publication


  • United Kingdom