Scrima, A. et al. Structural basis of UV DNA-damage recognition by the DDB1-DDB2 complex. Cell 135, 1213-1223

Friedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH 4058 Basel, Switzerland.
Cell (Impact Factor: 32.24). 01/2009; 135(7):1213-23. DOI: 10.1016/j.cell.2008.10.045
Source: PubMed


Ultraviolet (UV) light-induced pyrimidine photodimers are repaired by the nucleotide excision repair pathway. Photolesions have biophysical parameters closely resembling undamaged DNA, impeding discovery through damage surveillance proteins. The DDB1-DDB2 complex serves in the initial detection of UV lesions in vivo. Here we present the structures of the DDB1-DDB2 complex alone and bound to DNA containing either a 6-4 pyrimidine-pyrimidone photodimer (6-4PP) lesion or an abasic site. The structure shows that the lesion is held exclusively by the WD40 domain of DDB2. A DDB2 hairpin inserts into the minor groove, extrudes the photodimer into a binding pocket, and kinks the duplex by approximately 40 degrees. The tightly localized probing of the photolesions, combined with proofreading in the photodimer pocket, enables DDB2 to detect lesions refractory to detection by other damage surveillance proteins. The structure provides insights into damage recognition in chromatin and suggests a mechanism by which the DDB1-associated CUL4 ubiquitin ligase targets proteins surrounding the site of damage.

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    • "Consistently, our previous transgenic study demonstrated that, like tomato DDB1, the down-regulation of tomato CUL4 results in an elevated plastid level and overproduction of pigments (Wang et al., 2008). In theory , DDB1 could either directly dock a substrate protein and regulate its ubiquitination or recruit a substrate target via an additional adaptor (Li et al., 2006; Scrima et al., 2008). In the case of ubiquitination/degradation of GLK2, the former seems likely to be due to the positive result of BiFC analyses that are known to work for direct interactions mediating a re-construction of the YFP fluorescence activity (Fig. 4c). "
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    ABSTRACT: CULLIN4-RING ubiquitin ligases (CRL4s) as well as their targets are fundamental regulators functioning in many key developmental and stress responses in eukaryotes. In tomato (Solanum lycopersicum), molecular cloning has revealed that the underlying genes of natural spontaneous mutations high pigment 1 (hp1), high pigment 2 (hp2) and uniform ripening (u) encode UV-DAMAGED DNA BINDING PROTEIN 1 (DDB1), DE-ETIOLATED 1 (DET1) and GOLDEN 2-LIKE (GLK2), respectively. However, the molecular basis of the opposite actions of tomato GLK2 vs CUL4-DDB1-DET1 complex on regulating plastid level and fruit quality remains unknown. Here, we provide molecular evidence showing that the tomato GLK2 protein is a substrate of the CUL4-DDB1-DET1 ubiquitin ligase complex for the proteasome degradation. SlGLK2 is degraded by the ubiquitin-proteasome system, which is mainly determined by two lysine residues (K11 and K253). SlGLK2 associates with the CUL4-DDB1-DET1 E3 complex in plant cells. Genetically impairing CUL4, DDB1 or DET1 results in a retardation of SlGLK2 degradation by the 26S proteasome. These findings are relevant to the potential of nutrient accumulation in tomato fruit by mediating the plastid level and contribute to a deeper understanding of an important regulatory loop, linking protein turnover to gene regulation.
    New Phytologist 09/2015; DOI:10.1111/nph.13635 · 7.67 Impact Factor
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    • "nent system , which controls heparin and heparan sulfate acquisition and degradation in the human gut symbiont Bacteroides thetaiotaomicron ( pdb entry 4a2l ; Lowe et al . , 2012 ) , ( 2 ) a xyloglucanase from C . thermocellum ( pdb entry 2cn3 , Martinez - Fleites et al . , 2006 ) , and ( 3 ) a human DNA - damage binding protein ( pdb entry 3ei3 ; Scrima et al . , 2008 ) . We have performed homology modeling based on the first three hits of pGenThreader ( Figure 6 ) . In all three models the first 110 aa were removed . Two of the models ( Figures 6A , C ) show a propeller - like assembly reminiscent of a WD40 repeat . Although not being suitable for homology modeling , also the lower ranking hits prop"
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    ABSTRACT: The uncultivated ”Ca. Altiarchaeum hamiconexum” (formerly known as SM1 Euryarchaeon) carries highly specialized nano-grappling hooks (“hami”) on its cell surface. Until now little is known about the major protein forming these structured fibrous cell surface appendages, the genes involved or membrane anchoring of these filaments. These aspects were analyzed in depth in this study using environmental transcriptomics combined with imaging methods. Since a laboratory culture of this archaeon is not yet available, natural biofilm samples with high Ca. A. hamiconexum abundance were used for the entire analyses. The filamentous surface appendages spanned both membranes of the cell, which are composed of glycosyl-archaeol. The hami consisted of multiple copies of the same protein, the corresponding gene of which was identified via metagenome-mapped transcriptome analysis. The hamus subunit proteins, which are likely to self-assemble due to their predicted beta sheet topology, revealed no similiarity to known microbial flagella-, archaella-, fimbriae- or pili-proteins, but a high similarity to known S-layer proteins of the archaeal phylum at their N-terminal region (47-44% identity). Our results provide new insights into the structure of the unique hami and their major protein and indicate their divergent evolution with S-layer proteins.
    Frontiers in Microbiology 06/2015; 6. DOI:10.3389/fmicb.2015.00543 · 3.99 Impact Factor
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    • "There are two subpathways in eukaryotic NER: global genome NER (GG-NER) and transcription-coupled NER (TC-NER)2. In GG-NER, the XPE protein, which consists of the DDB1 and DDB2 subunits, recognizes the lesion by forming a specific complex with damaged DNA5, and then the DNA is transferred to the XPC protein, through the ubiquitylation of both proteins by the ubiquitin ligase bound to DDB16. After this damage recognition step, TFIIH, a general transcription factor, and the XPA protein are recruited. "
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    ABSTRACT: To maintain genetic integrity, ultraviolet light-induced photoproducts in DNA must be removed by the nucleotide excision repair (NER) pathway, which is initiated by damage recognition and dual incisions of the lesion-containing strand. We intended to detect the dual-incision step of cellular NER, by using a fluorescent probe. A 140-base pair linear duplex containing the (6-4) photoproduct and a fluorophore-quencher pair was prepared first. However, this type of DNA was found to be degraded rapidly by nucleases in cells. Next, a plasmid was used as a scaffold. In this case, the fluorophore and the quencher were attached to the same strand, and we expected that the dual-incision product containing them would be degraded in cells. At 3 h after transfection of HeLa cells with the plasmid-type probes, fluorescence emission was detected at the nuclei by fluorescence microscopy only when the probe contained the (6-4) photoproduct, and the results were confirmed by flow cytometry. Finally, XPA fibroblasts and the same cells expressing the XPA gene were transfected with the photoproduct-containing probe. Although the transfer of the probe into the cells was slow, fluorescence was detected depending on the NER ability of the cells.
    Scientific Reports 07/2014; 4:5578. DOI:10.1038/srep05578 · 5.58 Impact Factor
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