Kaoru Sugasawa

Xeroderma Pigmentosum Group C Protein Complex Is the Initiator of Global Genome Nucleotide Excision Repair

The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled nucleotide excision repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.

UV-Induced Ubiquitylation of XPC Protein Mediated by UV-DDB-Ubiquitin Ligase Complex

The xeroderma pigmentosum group C (XPC) protein complex plays a key role in recognizing DNA damage throughout the genome for mammalian nucleotide excision repair (NER). Ultraviolet light (UV)-damaged DNA binding protein (UV-DDB) is another complex that appears to be involved in the recognition of NER-inducing damage, although the precise role it plays and its relationship to XPC remain to be elucidated. Here we show that XPC undergoes reversible ubiquitylation upon UV irradiation of cells and that this depends on the presence of functional UV-DDB activity. XPC and UV-DDB were demonstrated to interact physically, and both are polyubiquitylated by the recombinant UV-DDB-ubiquitin ligase complex. The polyubiquitylation altered the DNA binding properties of XPC and UV-DDB and appeared to be required for cell-free NER of UV-induced (6-4) photoproducts specifically when UV-DDB was bound to the lesion. Our results strongly suggest that ubiquitylation plays a critical role in the transfer of the UV-induced lesion from UV-DDB to XPC.

Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23.

Complementation group C of xeroderma pigmentosum (XP) represents one of the most common forms of this cancer‐prone DNA repair syndrome. The primary defect is located in the subpathway of the nucleotide excision repair system, dealing with the removal of lesions from the non‐transcribing sequences (‘genome‐overall’ repair). Here we report the purification to homogeneity and subsequent cDNA cloning of a repair complex by in vitro complementation of the XP‐C defect in a cell‐free repair system containing UV‐damaged SV40 minichromosomes. The complex has a high affinity for ssDNA and consists of two tightly associated proteins of 125 and 58 kDa. The 125 kDa subunit is an N‐terminally extended version of previously reported XPCC gene product which is thought to represent the human homologue of the Saccharomyces cerevisiae repair gene RAD4. The 58 kDa species turned out to be a human homologue of yeast RAD23. Unexpectedly, a second human counterpart of RAD23 was identified. All RAD23 derivatives share a ubiquitin‐like N‐terminus. The nature of the XP‐C defect implies that the complex exerts a unique function in the genome‐overall repair pathway which is important for prevention of skin cancer.

Interaction of hHR23 with S5a THE UBIQUITIN-LIKE DOMAIN OF hHR23 MEDIATES INTERACTION WITH S5a SUBUNIT OF 26 S PROTEASOME

hHR23B is one of two human homologs of theSaccharomyces cerevisiae nucleotide excision repair (NER) gene product RAD23 and a component of a protein complex that specifically complements the NER defect of xeroderma pigmentosum group C (XP-C) cell extracts in vitro. Although a small proportion of hHR23B is tightly complexed with the XP-C responsible gene product, XPC protein, a vast majority exists as an XPC-free form, indicating that hHR23B has additional functions other than NER in vivo. Here we demonstrate that the human NER factor hHR23B as well as another human homolog of RAD23, hHR23A, interact specifically with S5a, a subunit of the human 26 S proteasome using the yeast two-hybrid system. Furthermore, hHR23 proteins were detected with S5a at the position where 26 S proteasome sediments in glycerol gradient centrifugation of HeLa S100 extracts. Intriguingly, hHR23B showed the inhibitory effect on the degradation of 125I-lysozyme in the rabbit reticulocyte lysate. hHR23 proteins thus appear to associate with 26 S proteasome in vivo. From co-precipitation experiments using several series of deletion mutants, we defined the domains in hHR23B and S5a that mediate this interaction. From these results, we propose that part of hHR23 proteins are involved in the proteolytic pathway in cells.

Crystal structure of thymine DNA glycosylase conjugated to SUMO-1.

Members of the small ubiquitin-like modifier (SUMO) family can be covalently attached to the lysine residue of a target protein through an enzymatic pathway similar to that used in ubiquitin conjugation, and are involved in various cellular events that do not rely on degradative signalling via the proteasome or lysosome. However, little is known about the molecular mechanisms of SUMO-modification-induced protein functional transfer. During DNA mismatch repair, SUMO conjugation of the uracil/thymine DNA glycosylase TDG promotes the release of TDG from the abasic (AP) site created after base excision, and coordinates its transfer to AP endonuclease 1, which catalyses the next step in the repair pathway. Here we report the crystal structure of the central region of human TDG conjugated to SUMO-1 at 2.1 Å resolution. The structure reveals a helix protruding from the protein surface, which presumably interferes with the product DNA and thus promotes the dissociation of TDG from the DNA molecule. This helix is formed by covalent and non-covalent contacts between TDG and SUMO-1. The non-covalent contacts are also essential for release from the product DNA, as verified by mutagenesis.

The Molecular Basis of CRL4DDB2/CSA Ubiquitin Ligase Architecture, Targeting, and Activation

The DDB1-CUL4-RBX1 (CRL4) ubiquitin ligase family regulates a diverse set of cellular pathways through dedicated substrate receptors (DCAFs). The DCAF DDB2 detects UV-induced pyrimidine dimers in the genome and facilitates nucleotide excision repair. We provide the molecular basis for DDB2 receptor-mediated cyclobutane pyrimidine dimer recognition in chromatin. The structures of the fully assembled DDB1-DDB2-CUL4A/B-RBX1 (CRL4(DDB2)) ligases reveal that the mobility of the ligase arm creates a defined ubiquitination zone around the damage, which precludes direct ligase activation by DNA lesions. Instead, the COP9 signalosome (CSN) mediates the CRL4(DDB2) inhibition in a CSN5 independent, nonenzymatic, fashion. In turn, CSN inhibition is relieved upon DNA damage binding to the DDB2 module within CSN-CRL4(DDB2). The Cockayne syndrome A DCAF complex crystal structure shows that CRL4(DCAF(WD40)) ligases share common architectural features. Our data support a general mechanism of ligase activation, which is induced by CSN displacement from CRL4(DCAF) on substrate binding to the DCAF.

Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein

ABSTRACT Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome nucleotide excision repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed α-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.

A molecular mechanism for DNA damage recognition by the xeroderma pigmentosum group C protein complex

The XPC-HR23B complex is involved in DNA damage recognition and the initiation of global genomic nucleotide excision repair (GG-NER). Our previous studies demonstrate that XPC-HR23B recognizes and binds DNA containing a helix distortion, regardless of the presence or absence of damaged bases. Here, we describe an extended analysis of the DNA binding specificity of XPC-HR23B using various defined DNA substrates. Although XPC-HR23B showed significantly higher affinity for single-stranded DNA than double-stranded DNA, specific secondary structures of DNA, involving a single- and double-strand junction, were strongly preferred by the complex. This indicates that the presence of bases, which cannot form normal Watson-Crick base pairs in double-stranded DNA, is a critical factor in determining the specificity of XPC-HR23B binding. A DNase I footprint analysis, using a looped DNA substrate, revealed that a single XPC-HR23B complex protected a distorted site in an asymmetrical manner, consistent with the preferred secondary structure. The specific binding of XPC-HR23B is undoubtedly an important molecular process, based on which NER machinery detects a wide variety of lesions that vary in terms of chemical structure during DNA repair.

Two human homologs of Rad23 are functionally interchangeable in complex formation and stimulation of XPC repair activity.

XPC-hHR23B protein complex is specifically involved in nucleotide excision repair (NER) of DNA lesions on transcriptionally inactive sequences as well as the nontranscribed strand of active genes. Here we demonstrate that not only highly purified recombinant hHR23B (rhHR23B) but also a second human homolog of the Saccharomyces cerevisiae Rad23 repair protein, hHR23A, stimulates the in vitro repair activity of recombinant human XPC (rhXPC), revealing functional redundancy between these human Rad23 homologs. Coprecipitation experiments with His-tagged rhHR23 as well as sedimentation velocity analysis showed that both rhHR23 proteins in vitro reconstitute a physical complex with rhXPC. Both complexes were more active than free rhXPC, indicating that complex assembly is required for the stimulation. rhHR23B was shown to stimulate an early stage of NER at or prior to incision. Furthermore, both rhHR23 proteins function in a defined NER system reconstituted with purified proteins, indicating direct involvement of hHR23 proteins in the DNA repair reaction via interaction with XPC.

XPC and Human Homologs of RAD23: Intracellular Localization and Relationship to Other Nucleotide Excision Repair Complexes

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus.