Abstract
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 high-density 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.
Highlights
In all cellular organisms, the genome is organized as a double stranded DNA helix with the nucleotide bases carrying the genetic information sequestered in the interior of the double strand, protected from damaging agents [1]
Using Alexa647-labelled Sulfolobus solfataricus SSB (SsoSSB) (A647SsoSSB) and Cy3-labelled single stranded DNA (ssDNA), we show that the close positioning of the labelled monomers on the ssDNA leads to a range of photophysical events including Forster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and acceptor quenching within the same single-molecule trajectory that can be exploited to dissect SSB dynamics
The majority of biophysical studies characterizing SSB proteins, at single-molecule level, have been carried out at room temperature [44], even for thermophilic species such as Thermus SSB [36]; we carried out a PIFE titration at 65◦C (Supplementary Figure S1), which is closer to the optimal growth temperature of S. solfataricus (75– 80◦C)
Summary
The genome is organized as a double stranded DNA helix (dsDNA) with the nucleotide bases carrying the genetic information sequestered in the interior of the double strand, protected from damaging agents [1]. To provide access to genomic information during vital cellular processes such as DNA replication, transcription, recombination and repair, dsDNA must be unwound to produce single stranded DNA (ssDNA) [2]. Transient exposure of the genetic information is crucial for cell survival, the presence of ssDNA intermediates increases the possibility of chemical and physical damage, compromising genome stability [2,3]. In addition to protecting ssDNA from degradation, the high affinity of SSBs has been exploited by nature to detect DNA damage [2, 5,6,7,8], melt dsDNA [5,6,7] and to recruit other proteins to ssDNA stimulating their biochemical activity and the overall efficiency of DNA processing pathways [9,10,11,12]. A number of studies have reported the use of SSBs, mostly of thermophilic origin due to their higher stability, to improve the efficiency and specificity of the polymerase chain reaction (PCR) [17, 18]
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