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. 2017 Oct 13;45(18):10555-10563.
doi: 10.1093/nar/gkx670.

Dynamic Stepwise Opening of Integron attC DNA Hairpins by SSB Prevents Toxicity and Ensures Functionality

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Free PMC article

Dynamic Stepwise Opening of Integron attC DNA Hairpins by SSB Prevents Toxicity and Ensures Functionality

Maj Svea Grieb et al. Nucleic Acids Res. .
Free PMC article

Abstract

Biologically functional DNA hairpins are found in archaea, prokaryotes and eukaryotes, playing essential roles in various DNA transactions. However, during DNA replication, hairpin formation can stall the polymerase and is therefore prevented by the single-stranded DNA binding protein (SSB). Here, we address the question how hairpins maintain their functional secondary structure despite SSB's presence. As a model hairpin, we used the recombinogenic form of the attC site, essential for capturing antibiotic-resistance genes in the integrons of bacteria. We found that attC hairpins have a conserved high GC-content near their apical loop that creates a dynamic equilibrium between attC fully opened by SSB and a partially structured attC-6-SSB complex. This complex is recognized by the recombinase IntI, which extrudes the hairpin upon binding while displacing SSB. We anticipate that this intriguing regulation mechanism using a base pair distribution to balance hairpin structure formation and genetic stability is key to the dissemination of antibiotic resistance genes among bacteria and might be conserved among other functional hairpins.

Figures

Figure 1.
Figure 1.
SSB requires a single-stranded DNA overhang to open the attC hairpin. (A) Schematic illustration of smFRET assay to study the SSB-IntI competition. The average distance between the donor (green) and acceptor (red) serves as a readout of the molecular conformation. (B) The secondary structure of the attCaadA7 hairpin predicted by RNAfold (43). (C) Binding of SSB to poly(dT)70. SSB binds in the SSB-65 binding mode at low protein-to-DNA ratios and in the SSB-35 binding mode at higher SSB concentrations (cSSB). (D) Inefficient SSB opening of the attC hairpin by SSB-65 and SSB-35 binding modes. (E) Increased hairpin opening efficiency due to the presence of a ssDNA overhang of 2, 3 or 9 dT nucleotides on the attC hairpin. (F) Hairpin opening efficiency quantified by SSB-35 proportion with ssDNA overhangs from 1 nt to 65 nt at cSSB = 1μM. DNA breathing was reduced in the attCGCclamp hairpin (grey), which hence requires a longer overhang for opening. (G) Hydrodynamic radius of SSB-ssDNA complexes at cSSB = 1 μM obtained by FCS. SSB binds to ssDNA as short as 7 nt and the binding affinity increases with the length of ssDNA until 17 nt. The green line presents the control without SSB. All error bars depict the 95% confidence interval.
Figure 2.
Figure 2.
SSB opens the attC hairpin in discrete steps. (A) Representative FRET time trace of the attCwt+3dT hairpin with 100 nM SSB. The state finding algorithm identifies four distinct FRET states highlighted in different colors. (B) Histogram of the found states, SSB-35 (cyan), SSB-65 (green), closed hairpin (red) and attC-6–SSB (dark blue) at cSSB = 100 nM. Inset: Schematic of the attC-6–SSB complex. (C) Histogram of attCAT. The attC-6–SSB and SSB-65 states are rarely populated. (D) Histogram of attCGConly. A dominant attC-6–SSB state is observed. (E) Summary of the extracted transition pathways and rates of SSB opening the attCwt+3dT hairpin in a free energy landscape diagram. Dashed line for cSSB = 1 nM, solid line for cSSB = 1 μM.
Figure 3.
Figure 3.
IntI outcompetes SSB for the attC hairpin. (A) The attC hairpin is labeled at the apical loop to minimize IntI-fluorophore interactions. When IntI binds to the hairpin, the EFRET shows the closed hairpin. The SSB-characteristic populations form in the presence of 1 μM SSB, summarized by the blue outline. Upon addition of IntI to the SSB-attC complex, the low EFRET peak appears, representing the displacement of SSB by IntI. (B) The same competition experiment with attCAT, which does not stabilize attC-6–SSB, characterized by the difference in the histograms at 1 μM SSB compared to the wild type. Here, SSB displacement by IntI is much less efficient. (C) In vivo recombination frequencies show that attCAT recombination is approximately 50% less efficient than the wildtype attC site. Error bars show the standard deviation (P = 0.093).
Figure 4.
Figure 4.
A conserved weighted basepair distribution allows for the dynamic SSB-hairpin equilibrium. (A) Analysis of the GC-content of 263 different attC hairpins obtained from the INTEGRALL database (12). attC sites were divided into a core region and apical region as depicted in cyan and green at the top. While the core region has a median GC-content (dashed line) of ≈50%, the apical region shows a median GC-content of ≈ 70%. The asterisks indicate a significant difference between the populations (P < 10−4). (B) Model of how SSB opens the attC hairpin via the attC-6–SSB state recognized by IntI.

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