Binding dynamics of a monomeric SSB protein to DNA : a single-molecule multi-process approach

People Programme of the European Union’s Seventh Framework Programme [REA 334496 to B.E.B.]; Leonardo da Vinci European Union Programme (to M.F.G.); Wellcome Trust [099149/Z/12/Z, 091825/Z/10/Z]. Funding for open access charge: Wellcome Trust; University of St Andrews.Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between highdensity binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.Publisher PDFPeer reviewe

On validation of multibody musculoskeletal models

We review the opportunities to validate multibody musculoskeletal models in view of the current transition of musculoskeletal modelling from a research topic to a practical simulation tool in product design, healthcare and other important applications. This transition creates a new need for justification that the models are adequate representations of the systems they simulate. The need for a consistent terminology and established standards is identified and knowledge from fields with a more progressed state-of-the-art in verification and validation is introduced. A number of practical steps for improvement of the validation of multibody musculoskeletal models are pointed out and directions for future research in the field are proposed. It is hoped that a more structured approach to model validation can help to improve the credibility of musculoskeletal models. </jats:p