The Structural Basis of DNA Binding by the Single-stranded DNA-binding Protein from Sulfolobus Solfataricus

Gamsjaeger R, et al., 465(2):337-46, Biochem J, 2015

Single-stranded DNA-binding proteins (SSBs) are a class of proteins that are present in all organisms. SSBs are known to have one or more characteristic conserved oligonucleotide/oligosaccharide-binding (OB) folds. Single stranded DNA (ssDNA) binds to these conserved OB domains with high affinity. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a single OB fold coupled to a flexible C-terminal tail. Recent studies have identified two new human SSBs with protein sequences that are similar to SsoSSB. Described herein is the determination of the structure of the OB domain of SsoSSB bound to ssDNA using NMR spectroscopy. SsoSSB mutational analysis was performed by Bio-Layer Interferometry (BLI) using a Pall ForteBio BLItz instrument. Streptavidin Biosensor tips were immobilized with 5' biotinylated ssDNA oligonucleotide (5'-AAATTTTTT-3'). The sensor tips were then exposed to serial dilutions of the protein. The KD value was calculated for the binding interaction. This study reveals that the ssDNA binding to SsoSSB is regulated by base-stacking of three key aromatic residues. In contrast, the human SSBs (RPA and hSSB1) involve only two aromatic residues. The results also demonstrate SsoSSB recognition of ssDNA with a footprint of five bases.

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