Abstract
Single-stranded DNA (ssDNA) is a product of many cellular processes that involve double-stranded DNA, for example during DNA replication and repair, and is formed transiently in many others. Measurement of ssDNA formation is fundamental for understanding many such processes. The availability of a fluorescent biosensor for the determination of single-stranded DNA provides an important route to achieve this. Single-stranded DNA binding proteins (SSBs) protect ssDNA from degradation, but can be displaced to allow processing of the ssDNA. Their tight binding of ssDNA means that they are very good candidates for the development of a biosensor. Previously, the single stranded DNA binding protein from Escherichia coli, labeled with a fluorophore, (DCC-EcSSB) was developed and used for this purpose. However, the multiple binding modes of this protein meant that interpretation of DCC-EcSSB fluorescence was potentially complex in terms of determining the amount of ssDNA. Here, we present an improved biosensor, developed using the tetrameric SSB from Plasmodium falciparum as a new scaffold for fluorophore attachment. Each subunit of this tetrameric SSB was labeled with a diethylaminocoumarin fluorophore at a single site on its surface, such that there is a very large, 20-fold, fluorescence increase when it binds to ssDNA. This adduct can be used as a biosensor to report ssDNA formation. Because SSB from this organism has a single mode of binding ssDNA, namely 65–70 bases per tetramer, over a wide range of conditions, the fluorescent SSB allows simple quantitation of ssDNA. The binding is fast, possibly diffusion controlled, and tight (dissociation constant for DCC-PfSSB <5 pM). Its suitability for real-time assays of ssDNA formation was demonstrated by measurement of AddAB helicase activity, unwinding double-stranded DNA.
Highlights
Many cellular processes involving DNA, such as replication, transcription and repair rely on separating double-stranded DNA to form single-stranded DNA
The highest fluorescence change was observed with PfSSB, in which C93 was labeled with IDCC (Table 1)
Labeling on the loop that was used with EcSSB (G26C in EcSSB aligns with G102C in PfSSB) gave relatively low responses
Summary
Many cellular processes involving DNA, such as replication, transcription and repair rely on separating double-stranded DNA (dsDNA) to form single-stranded DNA (ssDNA). Major differences occur in the intrinsically disordered region at the C-terminus, which is 65 amino acids long in EcSSB and >80 amino acids long in PfSSB [11, 12, 14, 15] This region is not visible in the crystal structures of either species’ SSB but includes the domain responsible for the cooperative binding of SSB to ssDNA and for the interaction with other proteins involved in the DNA replication [12, 16, 17]. The best combination of fluorophore, labeling position and protein construct was chosen, based on the magnitude of the fluorescence change on binding ssDNA and on the ability of the adduct to bind ssDNA in a single mode over a wide range of solution conditions. The ability of this adduct to monitor ssDNA formation in a real-time assay is confirmed by a helicase activity assay
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