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DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding - PubMed

DNA binding properties of human Cdc45 suggest a function as molecular wedge for DNA unwinding

Anna Szambowska et al. Nucleic Acids Res. 2014 Feb.

Abstract

The cell division cycle protein 45 (Cdc45) represents an essential replication factor that, together with the Mcm2-7 complex and the four subunits of GINS, forms the replicative DNA helicase in eukaryotes. Recombinant human Cdc45 (hCdc45) was structurally characterized and its DNA-binding properties were determined. Synchrotron radiation circular dichroism spectroscopy, dynamic light scattering, small-angle X-ray scattering and atomic force microscopy revealed that hCdc45 exists as an alpha-helical monomer and possesses a structure similar to its bacterial homolog RecJ. hCdc45 bound long (113-mer or 80-mer) single-stranded DNA fragments with a higher affinity than shorter ones (34-mer). hCdc45 displayed a preference for 3' protruding strands and bound tightly to single-strand/double-strand DNA junctions, such as those presented by Y-shaped DNA, bubbles and displacement loops, all of which appear transiently during the initiation of DNA replication. Collectively, our findings suggest that hCdc45 not only binds to but also slides on DNA with a 3'-5' polarity and, thereby acts as a molecular 'wedge' to initiate DNA strand displacement.

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Figures

Figure 1.
Figure 1.

hCdc45 represents a well-folded monomer in solution. (A) AFM imaging of hCdc45 reveals a homogeneous population of monomeric protein molecules in solution. The scale bar corresponds to 100 nm. The enlarged inset shows examples of elongated Cdc45 shapes. (B) AFM volume analysis indicates a molecular mass of 63 kDa for human Cdc45, consistent with a homogeneous population of monomeric protein molecules in solution. AFM volumes are translated into protein molecular weight based on a linear calibration curve (inset).

Figure 2.
Figure 2.

Synchrotron radiation circular dichroism spectrum of hCdc45. (A) The spectrum was recorded at the UV-CD12 beamline of the ANKA storage ring, Karlsruhe, Germany, in 10 mM potassium phosphate, pH 7.5, and 150 mM NaF. The ellipticities θ, in deg dmol-cm, is plotted as a function of wavelength. Data processing was carried out using the CDtool software. (B) The secondary structure elements of hCdc45 were assessed using the indicated algorithms and the SP175 reference dataset, available on the DichroWeb server. An asterisk indicates the normalized root-mean-square deviation between calculated and experimental spectra.

Figure 3.
Figure 3.

Three-dimensional structural models of hCdc45 protein. (A) Superposition of the GASBOR and DAMMIF models. (B) Superposition of the DAMMIF and BUNCH models. The ab initio bead model of hCdc45 in (A) and (B) is presented as transparent yellow spheres, the model from GASBOR as an orange surface, the model from BUNCH as a molecular surface with the N-terminal domain in green, the C-terminal domain in turquoise and the helical insertion or loop in magenta. The two figures are related by a rotation of 90° around the abscissa. (C) The crystal structure of T. thermophilus RecJ (1IR6) with the same coloring as in the BUNCH model for comparison. The BUNCH model is based on the N-and C-terminal domains of RecJ, whereas the gray part, corresponding to the linker and insertion, was rebuilt in the modeling. (D) SAXS curve of hCdc45. The experimental SAXS profile of human Cdc45 protein (black dots) is compared with the theoretical scattering curves calculated from the BUNCH model (red), the DAMMIF model (blue) and the RecJ crystal structure (1IR6) (green). (E) Guinier plot of hCdc45. The linearity of the Guiner plot (plotted in the range of 0.8 < sRg < 1.3) indicates the quality of the experimental SAXS data.

Figure 4.
Figure 4.

Binding of hCdc45 to ssDNA substrates. (A) Electrophoretic mobility shift assays were performed with 2 nM 5′-labeled ssDNA and the indicated amounts of hCdc45. After 10 min at 30°C, the reaction mixtures were cooled to 0°C. After addition of 5-µl loading dye, the samples were electrophoresed through a 10% non-denaturing polyacrylamide gels in 1× TBE buffer. Subsequently, gels were dried and exposed to a phosphorimaging screen. Gels were visualized using a phosphor-imager (Typhoon Trio; GE Healthcare). (B) The graph represents the percentage of bound DNA per added concentration of hCdc45 as calculated by the Image Quant software.

Figure 5.
Figure 5.

Binding of hCdc45 to different DNA structures. (A) Electrophoretic mobility shift assays were performed with 4 nM 5′-labeled DNA substrates and the indicated amounts of hCdc45. Gel electrophoresis was as described in the legend for Figure 4. The used oligonucleotides are listed in parentheses. The asterisks (*) represent the 5′-label. (B) Real-time binding analysis of various DNA substrates to hCdc45. Direct interaction between Cdc45 and DNA substrates was monitored using the BIAcore system. Binding was measured at 25°C at a flow rate of 30 µl/min in 20 mM HEPES-KOH, pH 7.5, 150 mM KCl, 1 mM DTT, 3 mM EDTA and the surfactant P20. The dissociation constants were calculated from the concentration-dependent steady-state response of DNA binding.

Figure 6.
Figure 6.

hCdc45 binding to branched DNA structures. (A) Electrophoretic mobility shift assays were performed using 2 nM 5′-labeled DNA substrates and the indicated amounts of hCdc45 as described in the legend for Figure 4. (B) The graph represents the percentage of unbound DNA as function of the hCdc45 concentration. The mean standard error from three independent experiments is also given. Densitometric analyses were performed using the Image-Quant software.

Figure 7.
Figure 7.

hCdc45 binds tightly to the 3′ arm of branched DNA structures. (A) hCdc45 binding to Y-shaped DNA and FLAP-DNA. Electrophoretic mobility shift assays were performed with 2-nM 5′-labeled DNA as described in the legend for Figure 4. (B) The graph represents the percentage of unbound DNA as function of the hCdc45 concentration with mean standard errors as described in the legend for Figure 6. (C) Binding of hCdc45 to 3′ and 5′-overhangs.

Figure 8.
Figure 8.

Hypothetical models for the action of hCdc45 on branched DNAs. (A) Binding of hCdc45 to ssDNA and sliding to the 5′ or 3′-end. (B) Preferred movement along ssDNA in the 3′–5′ direction aids to find the ss/ds junction (C) hCdc45 possesses a higher affinity for 5′-blocked flaps as compared with 3′-blocked structures. (D) Cdc45 may migrate in front of the Mcm2-7 helicase from 3′ to 5′ and, acting as a molecular ‘wedge’, to help displace the lagging strand.

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