Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans
Single‐stranded DNA binding protein (Ssb) ofDeinococcus radiodurans comprises N‐ and C‐terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N‐terminal without c...
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| Format: | Article |
| Language: | English |
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Wiley
2015-01-01
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| Series: | FEBS Open Bio |
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| Online Access: | https://doi.org/10.1016/j.fob.2015.04.009 |
| _version_ | 1811185123317514240 |
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| author | Aman K. Ujaoney Bhakti Basu K. Muniyappa Shree K. Apte |
| author_facet | Aman K. Ujaoney Bhakti Basu K. Muniyappa Shree K. Apte |
| author_sort | Aman K. Ujaoney |
| collection | DOAJ |
| description | Single‐stranded DNA binding protein (Ssb) ofDeinococcus radiodurans comprises N‐ and C‐terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N‐terminal without connector), SsbNC (N‐terminal with connector) and SsbC (C‐terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single‐stranded DNA (ssDNA) binding activity, compared to the full‐length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo‐dimers while SsbNC/SsbN formed different oligomeric forms.In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase‐I activity, but failed to stimulate Deinococcal RecA‐promoted DNA strand exchange. The results suggest that the C‐terminal OB fold is primarily responsible for ssDNA binding. The N‐terminal OB fold binds weakly to ssDNA but is involved in multimerization. |
| first_indexed | 2024-04-11T13:24:04Z |
| format | Article |
| id | doaj.art-ea0a724067514932b1c90def835c8c9b |
| institution | Directory Open Access Journal |
| issn | 2211-5463 |
| language | English |
| last_indexed | 2024-04-11T13:24:04Z |
| publishDate | 2015-01-01 |
| publisher | Wiley |
| record_format | Article |
| series | FEBS Open Bio |
| spelling | doaj.art-ea0a724067514932b1c90def835c8c9b2022-12-22T04:22:07ZengWileyFEBS Open Bio2211-54632015-01-015137838710.1016/j.fob.2015.04.009Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radioduransAman K. Ujaoney0Bhakti Basu1K. Muniyappa2Shree K. Apte3Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, IndiaMolecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, IndiaDepartment of Biochemistry, Indian Institute of Science, Bangalore 560012, IndiaMolecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, IndiaSingle‐stranded DNA binding protein (Ssb) ofDeinococcus radiodurans comprises N‐ and C‐terminal oligonucleotide/oligosaccharide binding (OB) folds connected by a beta hairpin connector. To assign functional roles to the individual OB folds, we generated three Ssb variants: SsbN (N‐terminal without connector), SsbNC (N‐terminal with connector) and SsbC (C‐terminal), each harboring one OB fold. Both SsbN and SsbNC displayed weak single‐stranded DNA (ssDNA) binding activity, compared to the full‐length Ssb (SsbFL). The level of ssDNA binding activity displayed by SsbC was intermediate between SsbFL and SsbN. SsbC and SsbFL predominantly existed as homo‐dimers while SsbNC/SsbN formed different oligomeric forms.In vitro, SsbNC or SsbN formed a binary complex with SsbC that displayed enhanced ssDNA binding activity. Unlike SsbFL, Ssb variants were able to differentially modulate topoisomerase‐I activity, but failed to stimulate Deinococcal RecA‐promoted DNA strand exchange. The results suggest that the C‐terminal OB fold is primarily responsible for ssDNA binding. The N‐terminal OB fold binds weakly to ssDNA but is involved in multimerization.https://doi.org/10.1016/j.fob.2015.04.009Deinococcus radioduransSsb proteinOB foldsEMSARecAStrand exchange |
| spellingShingle | Aman K. Ujaoney Bhakti Basu K. Muniyappa Shree K. Apte Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans FEBS Open Bio Deinococcus radiodurans Ssb protein OB folds EMSA RecA Strand exchange |
| title | Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans |
| title_full | Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans |
| title_fullStr | Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans |
| title_full_unstemmed | Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans |
| title_short | Functional roles of N‐terminal and C‐terminal domains in the overall activity of a novel single‐stranded DNA binding protein ofDeinococcus radiodurans |
| title_sort | functional roles of n terminal and c terminal domains in the overall activity of a novel single stranded dna binding protein ofdeinococcus radiodurans |
| topic | Deinococcus radiodurans Ssb protein OB folds EMSA RecA Strand exchange |
| url | https://doi.org/10.1016/j.fob.2015.04.009 |
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