title: Functional roles of N-terminal and C-terminal domains in the overall activity of a novel single-stranded DNA binding protein of Deinococcus radiodurans.
abstract: Single-stranded DNA binding protein (Ssb) of Deinococcus 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.
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, India.
authors.forename: Aman K
authors.initials: AK
authors.lastname: Ujaoney
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, India.
authors.forename: Bhakti
authors.initials: B
authors.lastname: Basu
authors.affiliation: Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
authors.forename: K
authors.initials: K
authors.lastname: Muniyappa
authors.affiliation: Molecular Biology Division, Bhabha Atomic Research Centre, Mumbai 400085, India.
authors.forename: Shree K
authors.initials: SK
authors.lastname: Apte
keywords: Deinococcus radiodurans; EMSA; ESDSA, extended synthesis-dependent strand annealing; OB fold, oligonucleotide/oligosaccharide binding fold; OB folds; RPA, Replication protein A; RecA; Ssb protein; Ssb, single-stranded DNA binding protein; SsbC, C-terminal Ssb; SsbN, N-terminal Ssb without connector; SsbNC, N-terminal Ssb with connector; Strand exchange; Topoisomerase activity; ssDNA, single-stranded DNA