Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

Specificity of Binding of Single-Stranded DNA-Binding Protein to Its Target

Single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA (ssDNA) and participate in all genetic processes involving ssDNA, such as replication, recombination, and repair. Here we applied atomic force microscopy to directly image SSB–DNA complexes under various conditions. We used the hybrid DNA construct methodology in which the ssDNA segment is conjugated to the DNA duplex. The duplex part of the construct plays the role of a marker, allowing unambiguous identification of specific and nonspecific SSB–DNA complexes. We designed hybrid DNA substrates with 5′- and 3′-ssDNA termini to clarify the role of ssDNA polarity on SSB loading. The hybrid substrates, in which two duplexes are connected with ssDNA, were the models for gapped DNA substrates. We demonstrated that <i>Escherichia coli</i> SSB binds to ssDNA ends and internal ssDNA regions with the same efficiency. However, the specific recognition by ssDNA requires the presence of Mg²⁺ cations or a high ionic strength. In the absence of Mg²⁺ cations and under low-salt conditions, the protein is capable of binding DNA duplexes. In addition, the number of interprotein interactions increases, resulting in the formation of clusters on double-stranded DNA. This finding suggests that the protein adopts different conformations depending on ionic strength, and specific recognition of ssDNA by SSB requires a high ionic strength or the presence of Mg²⁺ cations

Single-Molecule Force Spectroscopy Studies of APOBEC3A–Single-Stranded DNA Complexes

APOBEC3A (A3A) inhibits the replication of a range of viruses and transposons and might also play a role in carcinogenesis. It is a single-domain deaminase enzyme that interacts with single-stranded DNA (ssDNA) and converts cytidines to uridines within specific trinucleotide contexts. Although there is abundant information that describes the potential biological activities of A3A, the interplay between binding ssDNA and sequence-specific deaminase activity remains controversial. Using a single-molecule atomic force microscopy spectroscopy approach developed by Shlyakhtenko et al. [(2015) <i>Sci. Rep. 5</i>, 15648], we determine the stability of A3A in complex with different ssDNA sequences. We found that the strength of the complex is sequence-dependent, with more stable complexes formed with deaminase-specific sequences. A correlation between the deaminase activity of A3A and the complex strength was identified. The ssDNA binding properties of A3A and those for A3G are also compared and discussed

AFM images of A3A in complex with gap-DNA.

<p>A3A monomer and dimer complexes are labeled 1 and 2, respectively. Bar size, 100</p

Gallery of AFM images of A3A_E72A complexed with dsDNA (A) or ssDNA (B) regions of the gap-DNA substrate.

<p>Bar size, 30</p

Histograms for protein volume measurements for free A3Gctd (A) and A3A (B).

<p>Number of complexes analyzed were N = 173 and 148 for histograms (A) and (B), respectively. The mean volume values for monomers (1-mers; 28±12 nm³) and dimers (2-mers; 56±22 nm³) are indicated with arrows. See similar numbers for A3A in Fig. 3.</p

AFM images of A3A_E72A complexed with gap-DNA substrate.

<p>Specific complexes with A3A_E72A dimers and trimers are labeled 2 and 3, respectively. Bar size, 100 nm.</p

Gallery of AFM images of A3A complexed with ssDNA (A) or dsDNA (B) regions of the gap-DNA substrate.

<p>Bar size, 30</p

Results of the volume measurements of A3A complexed with gap-DNA (A) alone (B).

<p>Numbers of complexes analyzed are N = 118 for (A) and N = 148 for (B). The mean volume values (± SD) for monomers (1-mers; 33±16 nm³) and dimers (2-mers; 66±26 nm³) are indicated with arrows.</p