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Sci Rep
2016 Jan 22;6:21501. doi: 10.1038/srep21501.
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Structural and biochemical characterization of an RNA/DNA binding motif in the N-terminal domain of RecQ4 helicases.
Marino F
,
Mojumdar A
,
Zucchelli C
,
Bhardwaj A
,
Buratti E
,
Vindigni A
,
Musco G
,
Onesti S
.
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The RecQ4 helicase belongs to the ubiquitous RecQ family but its exact role in the cell is not completely understood. In addition to the helicase domain, RecQ4 has a unique N-terminal part that is essential for viability and is constituted by a region homologous to the yeast Sld2 replication initiation factor, followed by a cysteine-rich region, predicted to fold as a Zn knuckle. We carried out a structural and biochemical analysis of both the human and Xenopus laevis RecQ4 cysteine-rich regions, and showed by NMR spectroscopy that the Xenopus fragment indeed assumes the canonical Zn knuckle fold, whereas the human sequence remains unstructured, consistent with the mutation of one of the Zn ligands. Both the human and Xenopus Zn knuckles bind to a variety of nucleic acid substrates, with a mild preference for RNA. We also investigated the effect of a segment located upstream the Zn knuckle that is highly conserved and rich in positively charged and aromatic residues, partially overlapping with the C-terminus of the Sld2-like domain. In both the human and Xenopus proteins, the presence of this region strongly enhances binding to nucleic acids. These results reveal novel possible roles of RecQ4 in DNA replication and genome stability.
Figure 1. Analysis of RecQ4 N-terminal region.(A) Structural organization of human RecQ4: the extended Sld2 homology region (blue), the Zn knuckle (green), the helicase core (red) and the RecQ C-terminal domain (RQC, in yellow). The two black rectangles indicate the regions encompassed by the protein constructs made for this study (ZnK and UpZnK). (B) Details of the synthesised peptides (pep-xZnK, pep-hZnK) and recombinant protein fragments (xZnK, hZnK, xUpZnK, hUpZnK) analysed in the present study. (C) Sequence alignment between RecQ4 and the C-terminus of Sld2 proteins. The human and Xenopus sequences are coloured in blue and the residues of the two synthetic peptides pep-hZnK and pep-xZnK are in bold. Residues that are conserved in 6/8 sequences are highlighted in yellow and cyan in RecQ4 and Sld2 proteins, respectively. Residues such as R/K/H, D/E, S/P/T/C, A/G, Y/F/W, I/L/V/M and Q/N, are classified as conserved. Residues conserved across RecQ4 and Sld2 are indicated by red asterisks. Amino acids involved in Zn2+ coordination are indicated by pink crosses. For the Zn knuckle the secondary structure elements (α-helices as rods and β-strands as arrows) are based on the Xenopus NMR structure (PDB ID: 2MPJ), otherwise they are based on the PsiPred prediction (http://bioinf.cs.ucl.ac.uk/psipred/).
Figure 2. Spectroscopic studies of the peptides corresponding to the human and Xenopus Zn knuckles (pep-hZnK and pep-xZnK).(A) The CD spectra of the two peptides are characteristic of unfolded proteins (black and yellow, respectively). The addition of Zn2+ in equimolar amount, triggers the formation of secondary structures for the Xenopus peptide but not for the human one. (B) Left: 1D 1H NMR spectra of pep-hZnK in absence (black) and presence (blue) of Zn2+. Right: 1D 1H NMR spectra of pep-xZnK in absence (black) and presence (pink) of Zn2+. (C) Left: NMR bundle of the best 15 structures for the Xenopus Zn knuckle peptide (pep-xZnK). Right: Cartoon representation of one representative NMR structure. The Zn2+ metal ion is in purple and residues involved in Zn2+ coordination are depicted as ball-and-sticks.
Figure 3. Quantitative EMSA analysis of various DNA/RNA complexes with the recombinant fragments of human and Xenopus RecQ4 corresponding to the Zn knuckle alone (hZnK and xZnK, panels(A,B) and the Zn knuckle with the upstream conserved region (hUpZnK and xUpZnK, panels (C,D). (E) Example of gel shift assays for the various protein fragments using a forked RNA substrate. The assay was carried out with increasing concentrations of proteins (0–5 μM for the hZnK and xZnK; 0–1.28 μM for the hUpZnK and xUpZnK). Recombinant proteins include a 6His-GST tag. Each experiment was repeated at least three times to plot the binding curves. Errors were very small: for the sake of clarity error bars are not shown on the plots.
Figure 4. Role of the Zn ligands in nucleic acid binding.(A) Binding curves for wild type hUpZnk and mutants (m1: Cys403Ala/Asn406Ala; m2: His411Ala/Cys416Ala; m3: Asn406Cys) with ssDNA, forkDNA, ssRNA and forkRNA. Each experiment was repeated at least three times. (B) Summary of the site-directed mutants. (C) Binding of the hUpZnK fragment to forkDNA and forkRNA substrates, in the presence of increasing amount of EDTA. The presence of EDTA strongly affects binding to DNA substrates, while does not impair binding to RNA, although it changes the relative intensity of the shifted bands.
Figure 5. Putative mode of nucleic acid binding.(A) A detail of the Zn knuckle-RNA interactions of mouse Lin28 with let-7 microRNA21 (PDB code: 3TRZ), showing the critical role of aromatic residues stacking with the nucleic acid basis. (B) The Xenopus Zn knuckle is shown in the same orientation; for the sake of clarity, secondary structure elements are not depicted. The two conserved hydrophobic residues Phe616 and Trp624 are shown as ball-and-stick.
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