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. 2016 Sep 23;291(39):20779-86.
doi: 10.1074/jbc.M116.736223. Epub 2016 Aug 12.

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

Stefanie H Chen  1 , Rose T Byrne-Nash  2 , Michael M Cox  2
Affiliations

Escherichia coli RadD Protein Functionally Interacts with the Single-stranded DNA-binding Protein

Stefanie H Chen et al. J Biol Chem. .

Abstract

The bacterial single-stranded DNA binding protein (SSB) acts as an organizer of DNA repair complexes. The radD gene was recently identified as having an unspecified role in repair of radiation damage and, more specifically, DNA double-strand breaks. Purified RadD protein displays a DNA-independent ATPase activity. However, ATP hydrolytic rates are stimulated by SSB through its C terminus. The RadD and SSB proteins also directly interact in vivo in a yeast two-hybrid assay and in vitro through ammonium sulfate co-precipitation. Therefore, it is likely that the repair function of RadD is mediated through interaction with SSB at the site of damage.

Keywords: ATPase; DNA repair; SSB; helicase; protein-DNA interaction; protein-protein interaction; radiation biology.

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Figures

FIGURE 1.
FIGURE 1.
RadD is active in ATP hydrolysis. A, schematic of the primary sequence of RadD protein. The dark gray boxes show the sites of the conserved superfamily 2 helicase motifs, whereas the light gray box shows the position of the suspected zinc finger domain. B, RadD hydrolysis rates in the presence of the chloride (KCl), acetate (KOAc), or glutamate (K-Glu) anions. C, RadD hydrolysis activity with increasing concentrations of potassium chloride. D, RadD hydrolysis rates at different pH levels. MES buffer was used for pH < 7 and Tris acetate was used for pH ≥ 7. E, RadD hydrolysis rates at increasing concentrations of RadD. F, Michaelis-Menten curve for RadD hydrolysis in the presence of increasing ATP concentrations. G, ATP hydrolysis rates of wild type RadD protein versus the RadD K37R (KR) ATPase-deficient protein. Error bars indicate the S.D. of at least three experiments.
FIGURE 2.
FIGURE 2.
RadD and SSB interact. A, yeast two-hybrid vectors containing ssb and radD are able to drive binding to the activation domain, leading to growth on His−/Ade− SD plates. Empty vectors do not support growth. AD = activation domain, BD = binding domain. B, purified SSB protein is able to pull down purified RadD during precipitation in 15% ammonium sulfate. Pelleted proteins were resuspended and run on a 4–15% acrylamide gel. SSB alone precipitates well, but RadD alone does not. SSB lacking the C terminus (ΔC8) is unable to co-precipitate RadD.
FIGURE 3.
FIGURE 3.
SSB stimulates the ATP hydrolysis of RadD. A, rates of RadD ATP hydrolysis at varying concentrations of full-length SSB. B, rates of RadD ATP hydrolysis in the presence of RadD alone, compensating SSB storage buffer, SSB protein (50 μm), or SSBΔC8 protein (50 μm). C, rates of RadD ATP hydrolysis at varying concentrations of SSB tail peptide (SSB-Ct). D, rates of RadD ATP hydrolysis in the presence of buffer (none), wild type SSB tail peptide (C10), SSB tail peptide missing the final phenylalanine (ΔF), or scrambled tail sequence peptide (mix) at 30 μm. E, rates of RadD ATP hydrolysis at varying concentrations of ATP when RadD alone, full-length SSB (10 μm), or SSB tail peptide (10 μm) is present. F, fluorescence polarization of increasing concentrations of RadD with 0.5 nm fluorescein-labeled SSB tail peptide. Error bars indicate the S.D. of at least three experiments.
FIGURE 4.
FIGURE 4.
RadD interacts with DNA. A, Electrophoretic mobility shift of 5-fold dilution series of RadD incubated with poly-dT100 oligonucleotide and no nucleotide, ATP, ADP, or ATPγS (adenosine 5′-O-(thiotriphosphate); 5 mm). B, electrophoretic mobility shift of 5-fold dilution series of RadD K37R incubated with poly-dT100 oligonucleotide with or without ATP (5 mm). C, electrophoretic mobility shift of RadD incubated with poly-dT100 oligonucleotide with increasing concentrations of SSB tail peptide with or without ATP (5 mm). D, fluorescence polarization assay of increasing concentrations of RadD incubated with fluorescent poly-dT100 oligonucleotide. E, RadD ATP hydrolysis rates in the presence of various concentrations of M13mp18 circular single-stranded DNA (css) in the presence or absence of full-length SSB (10 μm).
FIGURE 5.
FIGURE 5.
Model of the role of RadD in response to DNA damage. A, a DNA lesion causes the RNA polymerase (RNAP) to stall, which in turn causes the replication fork to stall. B, SSB recruits RadD to the site of the stalled replisome. C, RadD removes RNA polymerase and stabilizes the revealed single-stranded DNA.
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