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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

How the tumor suppressor p53 recognizes its DNA response elements

In response to cellular stress signals, the tumor suppressor p53 acts as transcription factor by binding as a tetramer to a wide range of DNA sites, thereby activating numerous genes that are critical for cancer prevention (Vogelstein et al., 2000). Its main functional domain, the DNA binding core domain (p53DBD), is accompanied by structured tetramerisation domain and intrinsically disordered N- and C-termini. p53 binds specifically to double-stranded DNA response elements (REs) which are composed of two decameric half-sites of the general form RRRCWWGYYY (R = A, G; W = A, T; Y = C, T) (el-Deiry et al., 1992). Our previous studies on crystal structures of p53DBD in complexes with consensus DNA sites revealed non-canonical Hoogsteen (HG) base pairs at the A/T doublets within conserved CATG motifs at the half-site centers, flanked by GGG/CCC (Kitayner et al., 2010). In the HG geometry, the A bases are rotated around their glycosidic bonds by nearly 180 deg relative to that of the common Watson-Crick (WC) geometry, to form alternative hydrogen bonds (Fig. 1A). As a result of the HG geometry, the backbone atoms on opposite DNA strands are closer by ~2Ã… in the region of HG base pairs compared to that of WC base pairs, affecting the shape of the DNA helix. This finding led to the proposal that the unique DNA shape enhances the stabilization of the p53DBD-DNA complex (Kitayner et al., 2010).

To characterize the effect of DNA shape on p53-DNA interactions in terms of structural and biochemical aspects, we used a novel approach to ‘lock’ base-pairing into WC or HG geometry. The method relies on designing REs where the central A/T doublets are replaced by modified nucleotides in a way that avoids introduction of bulky groups and preserves the A/T patterns at the minor-groove edges of the modified base-pairs in both geometries. Thus, to shift the equilibrium toward the HG geometry, A bases were replaced by 2-oxo-A (2OA), and to enforce the WC geometry, A and T bases were replaced by Inosine (I) and 5-methyl-C (5mC), respectively (Figs. 1B,C). In this manner, the selection between the two forms, HG or WC, is determined by the balance between attractive and repulsive interactions, as manifested by the high-resolution crystal structures of the two types of p53-DNA complexes, displaying the expected conformational differences between the two helices (Figs. 2A,B). A detailed analysis of the various structures combined with DNA binding and kinetic studies demonstrated that complexes with the unusual HG geometry are stabilized relative to those with all-WC base pairs. We also showed that two natural high-affinity REs, related to DNA-damage response and incorporate CATG motifs, are also predisposed to adopt the unique DNA shape induced by HG base pairs (Golovenko et al., 2018).

To conclude, the combined structural and biochemical data demonstrate that in addition to direct readout made by direct interactions between the protein and DNA, indirect readout rendered by shape readout plays a major role in DNA recognition by proteins.

dima-fig1.gif

Figure 1. Schematic representation of Watson Crick and Hoogsteen base pairs for natural and modified variants. Hydrogen bonds between donor and acceptor groups indicated by blue bars, and repulsive interactions by red bars.

References

    el-Deiry, W. S., Kern, S. E., Pietenpol, J. A., Kinzler, K. W., & Vogelstein, B. (1992). Definition of a consensus binding site for p53. Nature Genetics, 1, 45-49.

    Golovenko, D., Brauning, B., Vyas, P., Haran, T. E., Rozenberg, H., & Shakked, Z. (2018). New Insights into the Role of DNA Shape on Its Recognition by p53 Proteins. Structure, 26, 1237-1250.

    Kitayner, M., Rozenberg, H., Rohs, R., Suad, O., Rabinovich, D., Honig, B., & Shakked, Z. (2010). Diversity in DNA recognition by p53 revealed by crystal structures with Hoogsteen base pairs. Nature Structural & Molecular Biology, 17, 423-429

    Vogelstein, B., Lane, D., & Levine, A. J. (2000). Surfing the p53 network. Nature, 408, 307-310.

Dmitrij Golovenko1,2
Oksana Degtjarik 1
Tali E. Haran 2
Haim Rozenberg1
Zippora Shakked1

1Department of Structural Biology
Weizmann Institute of Science
Rehovot 76100, Israel

2Department of Biology
Technion–Israel Institute of Technology
Haifa 32000, Israel

Ph: (972)-585-79-1784
E-mail: dmitrij.golovenko@gmail.com




































dima-fig2.gif

Figure 2. Structures of p53DBD tetramers bound to DNA REs (20 bp long) with 4 base pairs ‘locked’ into Hoogsteen geometry (A) or Watson-Crick geometry (B) (highlighted by thick lines).