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.
Highlights
Deinococcus radiodurans exhibits extreme resistance to radiation, desiccation and chemical mutagens [1,2,3]
Sequence similarity analyses of Deinococcal single-stranded DNA binding protein (Ssb) showed that N-terminal Ssb with connector (SsbNC) shares 33% identity and 51% similarity with Ssb-C-terminal domain (SsbC) and 38% identity and 49% similarity with E. coli Ssb (Blastp analysis, data not shown)
Deinococcus-Thermus group of bacteria, known to thrive in extreme environments that readily cause breach in DNA integrity, encode a novel Ssb composed of 2 asymmetric Oligosaccharide Binding (OB) folds linked by a 10 amino acid long connector
Summary
Deinococcus radiodurans exhibits extreme resistance to radiation, desiccation and chemical mutagens [1,2,3] This resistance is conferred by a variety of coping strategies adopted by the bacterial cell, such as high manganese to iron ratios, Extended Synthesis Dependent Strand Annealing (ESDSA), a condensed nucleoid structure and robust error-free DNA repair system [4,5,6]. The single-stranded DNA binding protein (Ssb) is crucial for all aspects of DNA metabolism [7]. It harbours Oligonucleotide/Oligosaccharide Binding (OB) folds which bind to ssDNA by electrostatic and base stacking interactions [8,9,10].
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