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(UV-DDB) Dimerization and Its Roles in Chromatinized DNA Repair

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(UV-DDB) Dimerization and Its Roles in Chromatinized DNA Repair Damaged DNA induced UV-damaged DNA-binding protein (UV-DDB) dimerization and its roles in chromatinized DNA repair Joanne I. Yeha,b,1, Arthur S. Levinec,d, Shoucheng Dua, Unmesh Chintea, Harshad Ghodkee, Hong Wangd,e, Haibin Shia, Ching L. Hsiehc,d, James F. Conwaya, Bennett Van Houtend,e, and Vesna Rapić-Otrinc,d aDepartments of Structural Biology, bBioengineering, cMicrobiology and Molecular Genetics, ePharmacology and Chemical Biology, and dUniversity of Pittsburgh Cancer Institute, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260 AUTHOR SUMMARY Exposure to UV radiation (DDB1-CUL4A DDB2) with can damage DNA that if chromatin modification and left unrepaired can cause the subsequent steps in the mutations leading to skin repair pathway. aging and skin cancer. In Here we report the crystal humans, the nucleotide structure of the full-length excision repair (NER) * human DDB2 bound to proteins function to damaged DNA in a complex recognize and repair with human DDB1 (Fig. P1). UV-damaged DNA. Defects While a large portion of the in DNA repair caused by N-terminal region of the mutations of these repair zebrafish DDB2 in the proteins have been linked earlier structure could not to several genetic diseases, be modeled, we have characterized by cancer resolved the 3D structure of predisposition (xeroderma the N-terminal domain of pigmentosum, XP) or DDB2. Our structure reveals premature aging (Cockayne secondary interactions syndrome), illustrating the between the N-terminal Fig. P1. Composite model of a dimeric DDB1-CUL4ADDB2 ubiquitin functional significance of DDB2 domain of DDB2 and a ligase-nucleosome complex. A model of a dimeric DDB1-CUL4A in a neighboring repair proteins to genomic complex with a nucleosome core particle, generated according to the relative integrity. UV-damaged subunit organization found in the dimeric UV-DDB-AP24 crystal structure. oligodeoxynucleotide DNA-binding protein The intermolecular orientation of the CUL4A-Rbx (in gray) subunit resulted molecule that substantially (UV-DDB) detects DNA from superimposing two copies of the respective BPA and BPC domains of augment the primary damage in the context of the DDB1 subunit in the DDB1-CUL4A-Rbx structure (accession number 2HYE) contacts formed by the nucleosomes (proteins onto the corresponding DDB1 domains (BPA in blue, BPC in green, BPB in β-barrel domain of DDB2. around which DNA is purple) in the dimeric UV-DDB crystal structure. The nucleosome core particle We propose that the packed in the nucleus), (PDB ID code 1AOI) was aligned by superpositioning the respective sugar- contacts formed between the phosphate backbones of the nucleosomal DNA (in blue) onto the backbones N-terminal domain of DDB2 initiating repair by of two adjacent AP24 oligodeoxynucleotides (not shown). Remarkably, the coordinating the damage available molecular volume between two AP24 DNA molecules and the damaged DNA recognition with the accommodates the spatial requirements of the nucleosome particle, with duplex trigger dimerization ubiquitin modification of minor adjustments of the two DDB2 subunits (in yellow). For visual clarity, of UV-DDB repair-associated proteins, the histones of the nucleosome core particle and the AP24 subunits are not ðDDB1-DDB2Þ2, leading to thus facilitating repair in depicted in this figure. For a view showing the superpositioning of the the formation of auxiliary the nucleus. nucleosome onto the two AP24 oligodeoxynucleotide molecules of the interactions between UV-DDB is composed of UV-DDB complex, refer to Fig. 4 of the complete on-line publication. UV-DDB and the DDB1 and DDB2 subunits, oligodeoxynucleotide that the latter of which is responsible for damaged DNA binding. Previous structural studies on a chimeric UV-DDB (human Author contributions: J.I.Y., A.S.L., B.V.H., and V.R.-O. designed research; J.I.Y., S.D., U.C., DDB1, zebrafish DDB2) found similar topological changes in H.G., H.W., H.S., C.L.H., and J.F.C. performed research; J.I.Y. contributed new damaged DNA regardless of the chemical nature of the lesion reagents/analytic tools; J.I.Y., S.D., U.C., H.G., H.W., H.S., J.F.C., B.V.H., and V.R.-O. analyzed (1). While this study provided a structural framework to define data; and J.I.Y., A.S.L., J.F.C., B.V.H., and V.R.-O. wrote the paper. the nature of intermolecular associations at the damaged The authors declare no conflict of interest. DNA lesion site, a number of key questions remained This article is a PNAS Direct Submission. unresolved. The specificity and the unprecedented damaged Freely available online through the PNAS open access option. DNA binding affinity found in UV-DDB, which is by several Data deposition: The atomic coordinates and structure factors have been deposited in the orders of magnitude greater than that of another DNA Protein Data Bank, www.pdb.org (PDB ID codes 4E54 and 4E5Z). damage discrimination factor in repair, the XPC-HR23B *Nucleotide excision repair (NER) proteins are referred to as “repair proteins” in the complex (2), remain unexplained. This study sought to gain summary. molecular insights into how DNA damage specificity is 1To whom correspondence should be addressed. E-mail: [email protected]. achieved and to link the ubiquitination reactions mediated by See full research article on page E2737 of www.pnas.org. the UV-DDB-containing cullin 4A E3 ligase complex Cite this Author Summary as: PNAS 10.1073/pnas.1110067109. 16408–16409 ∣ PNAS ∣ October 9, 2012 ∣ vol. 109 ∣ no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1110067109 Downloaded by guest on September 26, 2021 substantially enhance damaged DNA binding affinities and complexes enables interaction with various protein targets and PNAS PLUS further stabilize the dimeric state of UV-DDB. These increases the efficiency of ubiquitination has gained crystallographic results were further bolstered by several prominence (5). However, experimental evidence for the complementary approaches that included electron microscopy existence of dimeric substrate–receptor complexes has been (EM) and atomic force microscopy (AFM). The EM and largely missing. Our finding of the dimeric structure of DDB2 AFM data indicate that UV-DDB in solution is monomeric, provides experimental support for the hypothetical role of but binding to damaged DNA dramatically promotes the the DDB1-CUL4A DDB2 E3 ligase complex. We propose that dimerization of the complex ðDDB1-DDB2Þ2. The AFM data UV-DDB dimerization organizes the molecular scaffold to further demonstrated that the dimeric UV-DDB can align the various molecular components assembled at the simultaneously bind to two DNA duplexes, corroborating the lesion to coordinate detection and initiation of repair in the dimeric conformation seen in the crystal structure. Considered context of a nucleosome containing DNA damage, thus altogether, the consistency and agreement of data obtained regulating a wide spectrum of functions pertinent to NER from multiple experimental approaches support the central and ubiquitin-proteosome pathways. functional role of the N-terminal domain of DDB2 enhancing substrate-binding affinities and specificities, which in turn 1. Scrima A, et al. (2008) Structural basis of UV DNA-damage recognition by the further stabilize the dimeric state of UV-DDB in a cooperative DDB1-DDB2 complex. Cell 135:1213–1223. manner. 2. Alekseev S, et al. (2008) Cellular concentrations of DDB2 regulate dynamic binding of DDB2 DDB1 at UV-induced DNA damage. Mol Cell Biol 28:7402–7413. After binding to damaged DNA, the DDB1-CUL4A 2 3. Kapetanaki M, et al. (2006) The DDB1-CUL4ADDB ubiquitin ligase is deficient in E3 ligase targets several associated proteins for mono- or xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites. polyubiquitination (e.g., histone H2A, XPC and DDB2) (3, 4). Proc Natl Acad Sci USA 103:2588–2593. However, it is not known whether different conformations of 4. Takedachi A, et al. (2010) DDB2 complex-mediated ubiquitylation around DNA damage the E3 ligase are required to facilitate the attachment of is oppositely regulated by XPC and Ku and contributes to the recruitment of XPA. Mol Cell Biol 30:2708–2723. various forms of ubiquitin to these substrates. Recently, the 5. Bosu DR, Kipreos ET (2008) Cullin-RING ubiquitin ligases: Global regulation and activa- concept that dimerization of the cullin-based E3 ligase tion cycles. Cell Div 3:7–19. BIOCHEMISTRY Yeh et al. PNAS ∣ October 9, 2012 ∣ vol. 109 ∣ no. 41 ∣ 16409 Downloaded by guest on September 26, 2021.
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