Multiple DNA damage recognition factors involved in mammalian nucleotide excision repair.

The nucleotide excision repair (NER) subpathway operating throughout the mammalian genome is a versatile DNA repair system that can remove a wide variety of helix-distorting base lesions. This system contributes to prevention of blockage of DNA replication by the lesions, thereby suppressing mutagenesis and carcinogenesis. Therefore, it is of fundamental significance to understand how the huge genome can be surveyed for occurrence of a small number of lesions. Recent studies have revealed that this difficult task seems to be accomplished through sequential actions of multiple DNA damage recognition factors, including UV-DDB, XPC, and TFIIH. Notably, these factors adopt completely different strategies to recognize DNA damage. XPC detects disruption and/or destabilization of the base pairing, which ensures a broad spectrum of substrate specificity for global genome NER. In contrast, UV-DDB directly recognizes particular types of lesions, such as UV-induced photoproducts, thereby vitally recruiting XPC as well as further extending the substrate specificity. After DNA binding by XPC, moreover, the helicase activity associated with TFIIH scans a DNA strand to make a final search for the presence of aberrant chemical modifications of DNA. The combination of these different strategies makes a crucial contribution to simultaneously achieving efficiency, accuracy, and versatility of the entire repair system.

Fluorescence correlation spectroscopy of the binding of nucleotide excision repair protein XPC-hHr23B with DNA substrates.

The interaction of the nucleotide excision repair (NER) protein dimeric complex XPC-hHR23B, which is implicated in the DNA damage recognition step, with three Cy3.5 labeled 90-bp double-stranded DNA substrates (unmodified, with a central unpaired region, and cholesterol modified) and a 90-mer single-strand DNA was investigated in solution by fluorescence correlation spectroscopy. Autocorrelation functions obtained in the presence of an excess of protein show larger diffusion times (tau (d)) than for free DNA, indicating the presence of DNA-protein bound complexes. The fraction of DNA bound (theta), as a way to describe the percentage of protein bound to DNA, was directly estimated from FCS data. A significantly stronger binding capability for the cholesterol modified substrate (78% DNA bound) than for other double-stranded DNA substrates was observed, while the lowest affinity was found for the single-stranded DNA (27%). This is in accordance with a damage recognition role of the XPC protein. The similar affinity of XPC for undamaged and 'bubble' DNA substrates (58% and 55%, respectively) indicates that XPC does not specifically bind to this type of DNA substrate comprising a large (30-nt) central unpaired region.

The outcome of the canalith repositioning procedure for benign paroxysmal positional vertigo: are there any characteristic features of treatment failure cases?

To demonstrate the success rate of the canalith repositioning procedure (CRP) in our clinic and to establish any characteristic features of cases of treatment failure, we reviewed clinical records of 62 patients who were diagnosed with posterior semicircular canal-benign paroxysmal positional vertigo (BPPV) and treated with the CRP. The basic strategy of the CRP was to rotate the involved canal slowly in the plane of gravity so that free-floating materials could migrate into the utricle only once. After the procedure we instructed patients to keep their heads upright for 10 h and not to sleep on the affected ear for 2 weeks. After the initial treatment, successful results were obtained in 51 of the 62 patients (82.2%). After the second treatment, 56 patients (90.3%) experienced success. Six patients (9.7%) did not obtain resolution even after the second treatment. While 46 patients were diagnosed with idiopathic BPPV, in 16 patients a different diagnosis was determined (head injury in 7 patients, Ménière's disease in 2, vestibular neuritis in 2 and unilateral sensorineural hearing loss in 5). We categorized these 16 patients as having secondary BPPV. Patients with idiopathic BPPV showed a significantly higher success rate with CRP than those with secondary BPPV. Patients with secondary BPPV may have quantitatively or qualitatively different lesions than those with idiopathic BPPV.

Changes in electrovestibular brainstem responses after aminoglycoside intoxication in guinea pigs.

OBJECTIVE: To verify the usefulness of short-latency vestibular responses evoked by a combination of round window electrical stimulation and sinusoidal rotation (electrovestibular brainstem responses; EVBRs) as a new monitoring tool of the vestibular function in animal experiments. METHODS: EVBRs were obtained before, during, and after treatment with aminoglycosides, along with compound action potential (CAP) audiograms. The changes in EVBRs were compared with morphological changes observed by scanning electron microscopy. RESULTS: EVBR amplitudes did not change in the group of guinea pigs treated with amikacin, but markedly decreased in the streptomycin and gentamicin- treated groups. CAP audiograms indicated a significant threshold elevation in the amikacin group, a moderate elevation in the gentamicin group, and no change in the streptomycin group. Under scanning electron microscopy, the loss of the sensory hair cells observed in the cristae ampullares was slight to moderate in the amikacin group, moderate to severe in the streptomycin group, and severe in the gentamicin group. CONCLUSION: EVBRs reflect overall pathological changes undergone by vestibular hair cells, and support the vestibular specificity of EVBRs.

Stereoselective reactions. XXXIV. Enantioselective deprotonation of prochiral 4-substituted cyclohexanones using chiral bidentate lithium amides having a bulky group instead of a phenyl group on the chiral carbon.

Using chiral bidentate lithium amides having a bulky group instead of a phenyl group on the chiral carbon, enantioselective deprotonation of prochiral 4-substituted cyclohexanones in the presence of excess trimethylsilyl chloride was examined in THF in the absence and in the presence of HMPA. It is shown that enantioselectivity of the reactions decreases as the substituent on the chiral carbon of the chiral lithium amides and the substituent at the 4-position of cyclohexanones become reasonably bulky. An eight-membered cyclic transition state model is proposed for this deprotonation reaction.

Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro.

The XPC-HR23B complex, a mammalian factor specifically involved in global genomic nucleotide excision repair (NER) has been shown to bind various forms of damaged DNA and initiate DNA repair in cell-free reactions. To characterize the binding specificity of this factor in more detail, a method based on immunoprecipitation was developed to assess the relative affinity of XPC-HR23B for defined lesions on DNA. Here we show that XPC-HR23B preferentially binds to UV-induced (6-4) photoproducts (6-4PPs) as well as to cholesterol, but not to the cyclobutane pyrimidine dimer (CPD), 8-oxoguanine (8-oxo-G), O6-methylguanine (O6-Me-G), or a single mismatch. Human whole cell extracts could efficiently excise 6-4PPs and cholesterol in an XPC-HR23B-dependent manner, but not 8-oxo-G, O6-Me-G or mismatches. Thus, there was good correlation between the binding specificity of XPC-HR23B for certain types of lesion and the ability of human cell extracts to excise these lesions, supporting the model that XPC-HR23B initiates global genomic NER. Although, XPC-HR23B does not preferentially bind to CPDs, the excision of CPDs in human whole cell extracts was found to be absolutely dependent on XPC-HR23B, in agreement with the in vivo observation that CPDs are not removed from the global genome in XP-C mutant cells. These results suggest that, in addition to the excision repair pathway initiated by XPC-HR23B, there exists another sub-pathway for the global genomic NER that still requires XPC-HR23B but is not initiated by XPC-HR23B. Possible mechanisms will be discussed.

A multistep damage recognition mechanism for global genomic nucleotide excision repair.

A mammalian nucleotide excision repair (NER) factor, the XPC-HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC-HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC-HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC-HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC-HR23B binding, probably due to enhancement of the helix distortion, cell-free excision of such compound lesions was much more efficient than expected from the observed affinity for XPC-HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.

Centrosome protein centrin 2/caltractin 1 is part of the xeroderma pigmentosum group C complex that initiates global genome nucleotide excision repair.

Nucleotide excision repair (NER) is carried out by xeroderma pigmentosum (XP) factors. Before the excision reaction, DNA damage is recognized by a complex originally thought to contain the XP group C responsible gene product (XPC) and the human homologue of Rad23 B (HR23B). Here, we show that centrin 2/caltractin 1 (CEN2) is also a component of the XPC repair complex. We demonstrate that nearly all XPC complexes contain CEN2, that CEN2 interacts directly with XPC, and that CEN2, in cooperation with HR23B, stabilizes XPC, which stimulates XPC NER activity in vitro. CEN2 has been shown to play an important role in centrosome duplication. Thus, those findings suggest that the XPC-CEN2 interaction may reflect coupling of cell division and NER.

Novel functional interactions between nucleotide excision DNA repair proteins influencing the enzymatic activities of TFIIH, XPG, and ERCC1-XPF.

The multisubunit basal transcription factor IIH (TFIIH) has a dual involvement in nucleotide excision repair (NER) of a variety of DNA lesions, including UV-induced photoproducts, and RNA polymerase II transcription. In both processes, TFIIH is implicated with local DNA unwinding, which is attributed to its helicase subunits XPB and XPD. To further define the role of TFIIH in NER, functional interactions between TFIIH and other DNA repair proteins were analyzed. We show that the TFIIH-associated ATPase activity is stimulated by both XPA and the XPC-HR23B complex. However, while XPA promotes the ATPase activity specifically in the presence of damaged DNA, stimulation by XPC-HR23B is lesion independent. Furthermore, we reveal that TFIIH inhibits the structure-specific endonuclease activities of both XPG and ERCC1-XPF, responsible for the 3' and 5' incision in NER, respectively. The inhibition occurs in the absence of ATP and is reversed upon addition of ATP. These results point toward additional roles for TFIIH and ATP during NER distinct from a requirement for DNA unwinding in the regulation of the endonuclease activities of XPG and ERCC1-XPF.

Risk factors for neurological complications after acoustic neurinoma radiosurgery: refinement from further experiences.

PURPOSE: Further actuarial analyses of neurological complications were performed on a larger population treated by stereotactic radiosurgery at our institution, to establish the optimal treatment parameters. METHODS AND MATERIALS: Between June 1990 and September 1998, 138 patients with acoustic neurinomas underwent stereotactic radiosurgery at Tokyo University Hospital. Of these, 125 patients who received medical follow-up for 6 months or more entered the present study. Patient ages ranged from 13 to 77 years (median, 53 years). Average tumor diameter ranged from 6.7 to 25.4 mm (mean, 13. 9 mm). Maximum tumor doses ranged from 20 to 40 Gy (mean, 29.8 Gy) and peripheral doses from 12 to 25 Gy (mean, 15.4 Gy). One to 12 isocenters were used (median, 4). Follow-up period ranged from 6 to 104 months (median, 37 months). The potential risk factors for neurological complications were analyzed by two univariate and one multivariate actuarial analyses. Neurological complications examined include hearing loss, facial palsy, and trigeminal nerve dysfunction. Variables included in the analyses were four demographic variables, two variables concerning tumor dimensions, and four variables concerning treatment parameters. A variable with significant p values (p < 0.05) on all three actuarial analyses was considered a risk factor. RESULTS: The variables that had significant correlation to increasing the risk for each neurological complication were: Neurofibromatosis Type 2 (NF2) for both total hearing loss and pure tone threshold (PTA) elevation; history of prior surgical resection, tumor size, and the peripheral tumor dose for facial palsy; and the peripheral tumor dose and gender (being female) for trigeminal neuropathy. In facial palsies caused by radiosurgery, discrepancy between the course of palsy and electrophysiological responses was noted. CONCLUSION: Risk factors for neurological complications seem to have been almost established, without large differences between institutions treating a large number of patients by radiosurgery. Radiosurgical doses and tumor dimensions were considered the two important risk factors for the 7th and 5th nerve injuries. Neurofibromatosis Type 2 was an important factor for hearing loss.

Retinoblastoma susceptibility protein, Rb, possesses multiple BRCT-Ws, BRCA1 carboxyl-terminus-related W regions with DNA break-binding activity.

The BRCT region, the carboxyl-terminus of BRCA1 (the breast cancer susceptibility gene 1 product), is ubiquitous in several proteins that participate in cell cycle checkpoints and DNA repair. We have previously shown that the BRCT regions of TopBP1 (DNA topoisomerase II binding protein 1) and BRCA1 bound DNA breaks. A BRCT-related region, BRCT-W1, in the retinoblastoma susceptibility gene product (Rb) also could bind DNA fragments, independently of DNA sequences. Five BRCT-W regions were found in the Rb family. All BRCT-Ws of Rb bound DNA fragments. Electron microscopy and treatment with an exonuclease showed that BRCT-Ws bound double-strand DNA breaks. Since some BRCTs are exceptional common relating elements in tumor suppression, our findings reveal novel aspects of the tumor suppression mechanism.

The xeroderma pigmentosum group C protein complex XPC-HR23B plays an important role in the recruitment of transcription factor IIH to damaged DNA.

The xeroderma pigmentosum group C protein complex XPC-HR23B was first isolated as a factor that complemented nucleotide excision repair defects of XP-C cell extracts in vitro. Recent studies have revealed that this protein complex plays an important role in the early steps of global genome nucleotide excision repair, especially in damage recognition, open complex formation, and repair protein complex formation. However, the precise function of XPC-HR23B in global genome repair is still unclear. Here we demonstrate that XPC-HR23B interacts with general transcription factor IIH (TFIIH) both in vivo and in vitro. This interaction is thought to be mediated through the specific affinity of XPC for the TFIIH subunits XPB and/or p62, which are essential for both basal transcription and nucleotide excision repair. Interestingly, association of TFIIH with DNA was observed in both wild-type and XP-A cell extracts but not in XP-C cell extracts, and XPC-HR23B could restore the association of TFIIH with DNA in XP-C cell extracts. Moreover, we found that XPC-HR23B was necessary for efficient association of TFIIH with damaged DNA in cell-free extracts. We conclude that the XPC-HR23B protein complex plays a crucial role in the recruitment of TFIIH to damaged DNA in global genome repair.

[Effects of corticosteroid on porcine retinal pigment epithelial cells in culture--2. Effects on phagocytosis and lysosomal activity].

PURPOSE: The effects of a corticosteoid on the phagocytosis and lysosomal activity of cultured porcine retinal pigment epithelium (RPE) cells were investigated. METHODS: After exposing cultured RPE cells to various concentrations (10, 50, 100, 500, 1,000 nM) of betamethasone sodium phosphate (betamethasone), the cells were incubated with latex microspheres for 6 hours. RESULTS: The number of latex microspheres phagocytized by the cultured RPE cells was inhibited by 50 nM betamethasone within 24 hours. Ten-nM betamethasone did not inhibit the proliferation of cultured RPE cells, but ingestion of latex microspheres by the cells was inhibited after 3 days. Lysosomal activity (acid phosphatase, beta-glucuronidase) of RPE cells was inhibited by a high concentration (500 nM) of betametasone. CONCLUSION: These results suggest that corticosteroid inhibits the phagocytosis and lysosomal activity of cultured RPE cells.

Reconstitution of damage DNA excision reaction from SV40 minichromosomes with purified nucleotide excision repair proteins.

We previously constructed the cell-free nucleotide excision repair (NER) assay system with UV-irradiated SV40 minichromosomes to analyze the mechanism of NER reaction on chromatin DNA. Here we investigate the factor that acts especially on nucleosomal DNA during the damage excision reaction, and reconstitute the damage excision reaction on SV40 minichromosomes. NER-proficient HeLa whole cell extracts were fractionated, and the amounts of known NER factors involved in the column fractions were determined by immunoblot analyses. The column fractions were quantitatively and systematically replaced by highly purified NER factors. Finally, damage DNA excision reaction on SV40 minichromosomes was reconstituted with six highly purified NER factors, XPA, XPC-HR23B, XPF-ERCC1, XPG, RPA and TFIIH, as those essential for the reaction with naked DNA. Further analysis showed that the damages on chromosomal DNA were excised as the same efficiency as those on naked DNA for short incubation. At longer incubation time, however, the damage excision efficiency on nucleosomal DNA was decreased whereas naked DNA was still vigorously repaired. These observations suggest that although the six purified NER factors have a potential to eliminate the damage DNA from SV40 minichromosomes, the chromatin structure may still have some repressive effects on NER.

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 the Saccharomyces 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 (125)I-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.

[Effects of corticosteroid on porcine retinal pigment epithelial cells in culture--1. Inhibitory effect on cell proliferation].

PURPOSE: Proliferation is an important function of the retinal pigment epithelial (RPE) cells. The effect of a corticosteroid on the proliferation of cultured porcine RPE cells was investigated. METHODS: After administration of various concentrations (10-1,000 nM) of betamethasone sodium phosphate (betamethasone), we counted RPE cell numbers at 1, 3, 6, 9, and 14 days. RESULTS: Betamethasone administration resulted in a dose-dependent decrease in RPE cell proliferation. The proliferation of the cultured RPE cells was significantly inhibited by betamethasone, at a dose of 300 nM in 9 days. Ten-nM betamethasone neither inhibited nor promoted the RPE cell proliferation in culture. CONCLUSIONS: These results suggest that corticosteroids inhibit proliferation of cultured porcine RPE cells.

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.

DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair.

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3'- or 5'-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5' side of its binding region, a weak ssDNA-binding domain resides at the 3' side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1-XPF on the DNA. With the 3'-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1-XPF, whereas the 5'-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.