Biological context

SSB proteins are involved in virtually all DNA processing events, including DNA repair (Richard et al. 2009). When DNA is damaged, SSBs detect this damage, protect the single-stranded DNA (ssDNA) that has been generated, and orchestrate repair. The importance of the SSB family in DNA processes is reflected by its evolutionary conservation. Bacterial and crenarchaeal SSBs have a simple domain organisation with one oligonucleotide binding (OB) fold (five-stranded bent antiparallel β-sheet that forms a closed β-barrel) followed by divergent spacer region and a charged C-terminus, whereas human replication protein A (RPA), the most widely studied SSB in humans, contains multiple OB domains in three different subunits (Iftode et al. 1999).

We have recently solved the three dimensional structure of the SSB from the crenarchaeote Sulfolobus solfataricus (SsoSSB) in solution alone, and in complex with ssDNA (Gamsjaeger et al. 2014, 2015) and discovered that it can detect DNA damage and recruit repair proteins (Cubeddu and White 2005). Mining the human database using the SsoSSB protein sequence as a template directly led us to discover two new SSBs: hSSB1 and hSSB2 (Richard et al. 2008) that both have a simple domain organisation. Over the last 8 years, we have contributed to the extensive characterisation of the biological function of hSSB1 (NABP2/OBFC2B) in DNA metabolism and have revealed that it plays a major role in the double stranded DNA break (DSB) repair pathway (Richard et al. 2008), in the response to oxidative DNA damage (Paquet et al. 2015), in the stability and repair of stalled DNA replication forks (Bolderson et al. 2014) and in maintenance of telomeres (Pandita et al. 2015).

The structure of the OB domain of hSSB1 bound to ssDNA was recently solved by X-ray crystallography as part of the SOSS1 complex, which consists of hSSB1, Ints3 and C9orf80, and plays a major role in DSB repair (Ren et al. 2014). Surprisingly, despite the high structural (RMSD 0.82 Å) and sequence (~55 %) similarity between SsoSSB and hSSB1, significant differences between the two ssDNA bound structures exist. For example, the crystal structure revealed that ssDNA recognition of hSSB1 is achieved via base-stacking of two essential aromatic residues with the ssDNA, rather than three, as observed in the SsoSSB-ssDNA structure (Gamsjaeger et al. 2015) and despite the fact that the third aromatic residue is conserved (W75 in SsoSSB, Y74 in hSSB1). However, due the lack of any structural information about ssDNA recognition by hSSB1 under more physiological conditions in solution, these discrepancies have so far not been resolved.

In this study, we present the solution-state backbone resonance assignments of hSSB1 at 298 K. We have also determined the DNA-binding interface in solution (by HSQC titration of 15N-labeled hSSB1 with 6T ssDNA); this interface is significantly different to the one in the existing crystal structure. These data in combination with further biophysical and biochemical experiments will allow us to elucidate the detailed atomic level mechanism of the interaction of hSSB1 with ssDNA under physiological conditions.

Methods and experiments

Protein expression and purification

Initially, an hSSB1 OB domain construct (1–123) was directionally cloned into pGEX-6P (with a GST expression tag) using the restriction enzymes BamHI and EcoRI. Protein expression using the E. coli BL21(DE3) strain was induced by addition of 0.2 mM IPTG at 25 °C for 16 h. Cells were lysed by sonication in 10 mM MES, pH 6.0, 50 mM NaCl, 3 mM TCEP, 0.5 mM PMSF, 0.1 % Triton X-100. Following centrifugation, the supernatant was subjected to GSH affinity chromatography followed by HRV-3C protease cleavage overnight at 4 °C (leaving the 5-residue stretch GPLGS at the N-terminus of the OB domain). The solution was applied to a HiTrap HP Heparin (2 × 5 mL tandem, GE) column equilibrated with NMR buffer (10 mM MES, pH 6.0, 50 mM NaCl, 3 mM TCEP). A 500 mL linear gradient comprising 50–1000 mM NaCl was used to elute cationic proteins. Fractions corresponding to a distinct absorbance peak were analysed by SDS-PAGE, pooled, concentrated and loaded onto a Superdex-75 gel filtration column in NMR buffer. 15N- and 15N13C-labeled hSSB1 protein was prepared using the procedure of (Cai et al. 1998) in a 5-L biofermentor and purified as described above. Protein concentrations were determined using the absorbance at 280 nm and the theoretical molar extinction coefficient for hSSB1.

NMR spectroscopy and data processing

NMR experiments were carried out using 0.2–0.8 mM hSSB1 in NMR buffer with 10 % D2O. Proton chemical shifts were referenced to 4,4-dimethyl-4-silapentanesulfonic acid (DSS) at 0 ppm. 13C and 15N chemical shifts were referenced indirectly to the same signal. NMR experiments were recorded at 298 K on either a Bruker 600 or 800 MHz spectrometer (Bruker Avance III) equipped with 5-mm TCI cryoprobes. The spectra recorded included 15N-HSQC, 13C-HSQC, CBCA(CO)NH, HNCACB, HNCO, HN(CA)CO and 15N-NOESY. The chemical shifts of DNA-bound 15N13C hSSB1 were unambiguously determined by stepwise addition of 0.2, 0.4, 0.6, 0.8 and 1 equimolar amounts of 6T ssDNA (HPLC purified; purchased from Sigma-Aldrich). Data were processed using Topspin (Bruker Biospin) and assignments were made using Sparky (T. D. Goddard and D. G. Kneller, University of California at San Francisco).

Assignments and data deposition

The hSSB1 construct used in this study contains the entire OB domain (1–123) as well as five additional residues from HRV-3C cleavage (see methods and experiments). Figure 1 shows the 15N HSQC spectrum of this folded protein domain. All backbone chain atoms with the exception of V105 and all prolines were assigned. The chemical shifts of the hSSB1 OB domain have been deposited in the BioMagResBank (http://www.bmrb.wisc.edu) under the accession number 26752.

Fig. 1
figure 1

15N-HSQC spectrum of hSSB1 OB domain (1–123 with five additional residues from HRV-3C cleavage, ~0.5 mM) showing backbone amide resonances. Note that for clarity, residues L-2, M1, N18, L19, I67, Q93, N112 and Q119 located in the centre of the spectrum are not labelled. The spectrum was recorded at a proton resonance frequency of 800 MHz at 298 K in 10 mM MES, pH 6.0, 50 mM NaCl, 3 mM TCEP

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In addition, we have mapped the DNA-binding interface of hSSB1 in solution onto the existing crystal structure (Fig. 2). Figure 2a depicts a portion of a 15N-HSQC spectrum of 15N-labeled hSSB1 in the presence (red) and absence (black) of ssDNA consisting of six thymines (6T). The recently published crystal structure of hSSB1 bound to ssDNA (PDB ID 4OWX) (Ren et al. 2014) has shown that six bases are sufficient for full binding. Calculation of weighted chemical shift changes (Ayed et al. 2001) upon binding to ssDNA (Fig. 2b) and mapping these changes onto the existing structure reveals the ssDNA recognition surface in solution (Fig. 2c, d, bound ssDNA coloured in green).

Fig. 2
figure 2

a Section of the 15N HSQC spectrum of ~0.3 mM hSSB1 alone (black) and a 1:1 mixture of hSSB1 and ssDNA (6T, red). Assignments and directions of movement are indicated. b Weighted backbone chemical shift changes of HN and N, (Ayed et al. 2001) for hSSB1 upon binding to ssDNA. Residues exhibiting changes larger than the average (solution binding residues) are coloured in salmon. Cartoon (c) and space-filling representation (d) of the crystal structure of hSSB1 bound to ssDNA (PDB 4OWX) (Ren et al. 2014) with solution binding residues and ssDNA coloured in salmon (in analogy to b) and green, respectively. Note that the DNA-binding interface observed in the crystal structure (PDB ID 4OWX) does not fully correlate with the observed chemical shift perturbations in solution. For example, G27, R28, T30, K31 and the aromatic residue Y85 (indicated by arrows in d) are not involved in DNA-binding in the crystal but exhibit significant chemical shift changes

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Intriguingly, comparison of the published crystal structure with our solution data uncovered five residues (G27, R28, T30, K31 and Y85, Fig. 2d) that exhibit substantial chemical shift perturbations in NMR HSQC experiments (Fig. 2b) but are not directly involved in DNA-binding in the crystal lattice. Further, the chemical shift change profile obtained for hSSB1 upon DNA recognition (Fig. 2b) closely resembled that measured for the structurally related SsoSSB (RMSD 0.82Å) (Fig. 2b in Gamsjaeger et al. 2014). The OB domain of SsoSSB also utilises a stretch of residues for DNA-binding (I30, Q31, T32, N34 and W75) that are structurally homologous to the five above-mentioned residues (Gamsjaeger et al. 2014, 2015). A possible explanation for these discrepancies is the presence of non-native crystal contacts between the ssDNA and the complex subunit, SOSSA, which the authors describe in their study (Ren et al. 2014). These artificial contacts have most likely distorted the configuration of the ssDNA in the crystal lattice. A future study using our NMR data, as well as further biophysical and biochemical experiments, will address these issues and provide the exact molecular details of DNA-binding by hSSB1 under physiological conditions.