Gene/Protein Disease Symptom Drug Enzyme Compound
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Studies of ultraviolet (UV) light mutagenesis have demonstrated mutations at common sites in the target genes of shuttle vector plasmids replicated in cultured cells or by cellular extracts. The reasons for the specific pattern of mutagenesis are largely unknown. We have examined the specificity of UV-induced mutagenesis by replicating plasmid pLS189, irradiated with 40 J/m(2) UVC or unirradiated, in either xeroderma pigmentosum group A (XP-A) or HeLa cellular extracts. The XP-A extract displayed slightly lower replication ability, but produced a higher mutant frequency, compared to that of HeLa extract. Use of irradiated plasmid inhibited replication by an average of 63% and increased the mutant frequency by an average of 16.7-fold. Analysis of mutation spectra revealed nonrandom patterns of mutagenesis that differed significantly between HeLa and XP-A extracts. In comparison to HeLa extract, replication in XP-A extract resulted in lower frequencies of GC --> AT transitions and tandem double-base substitutions, and a higher frequency of deletions. Replication in HeLa extract produced hotspots at positions 100, 108, and 156 that were not produced by XP-A extract. Furthermore, XP-A extract produced hotspots at positions 124, 133, and 164, sites not characteristic of previous UV-induced mutagenesis studies using XPA-expressing cells. Addition of purified XPA protein to reactions containing XP-A extract altered each of these parameters, including loss of the hotspots at positions 124 and 133, to yield a more HeLa-like spectrum. These results indicate a previously uncharacterized role of the XPA protein in influencing the specificity of UV-induced mutagenesis during DNA replication.
Environ Mol Mutagen 2001
PMID:XPA protein alters the specificity of ultraviolet light-induced mutagenesis in vitro. 1142 83

Cellular genomes are vulnerable to an array of DNA-damaging agents, of both endogenous and environmental origin. Such damage occurs at a frequency too high to be compatible with life. As a result cell death and tissue degeneration, aging and cancer are caused. To avoid this and in order for the genome to be reproduced, these damages must be corrected efficiently by DNA repair mechanisms. Eukaryotic cells have multiple mechanisms for the repair of damaged DNA. These repair systems in humans protect the genome by repairing modified bases, DNA adducts, crosslinks and double-strand breaks. The lesions in DNA are eliminated by mechanisms such as direct reversal, base excision and nucleotide excision. The base excision repair eliminates single damaged-base residues by the action of specialized DNA glycosylases and AP endonucleases. Nucleotide excision repair excises damage within oligomers that are 25 to 32 nucleotides long. This repair utilizes many proteins to remove the major UV-induced photoproducts from DNA, as well as other types of modified nucleotides. Different DNA polymerases and ligases are utilized to complete the separate pathways. The double-strand breaks in DNA are repaired by mechanisms that involve DNA protein kinase and recombination proteins. The defect in one of the repair protein results in three rare recessive syndromes: xeroderma pigmentosum, Cockayne syndrome, and trichothiodystrophy. This review describes the biochemistry of various repair processes and summarizes the clinical features and molecular mechanisms underlying these disorders.
Crit Rev Biochem Mol Biol 2001
PMID:Unraveling DNA repair in human: molecular mechanisms and consequences of repair defect. 1145 Sep 71

Here, we describe the assembly of the nucleotide excision repair (NER) complex in normal and repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of repair proteins at the site of the damage rather than a preassembled repairosome.
Mol Cell 2001 Jul
PMID:Sequential assembly of the nucleotide excision repair factors in vivo. 1151 74

The damaged-DNA binding protein DDB consists of two subunits, DDB1 (127 kDa) and DDB2 (48 kDa). Mutations in the DDB2 subunit have been detected in patients suffering from the repair deficiency disease xeroderma pigmentosum (group E). In addition, recent studies suggested a role for DDB2 in global genomic repair. DDB2 also exhibits transcriptional activity. We showed that expression of DDB1 and DDB2 stimulated the activity of the cell cycle regulatory transcription factor E2F1. Here we show that DDB2 is a cell cycle-regulated protein. It is present at a low level in growth-arrested primary fibroblasts, and after release the level peaks at the G(1)/S boundary. The cell cycle regulation of DDB2 involves posttranscriptional mechanisms. Moreover, we find that an inhibitor of 26S proteasome increases the level of DDB2, suggesting that it is regulated by the ubiquitin-proteasome pathway. Our previous study indicated that the cullin family protein Cul-4A associates with the DDB2 subunit. Because cullins are involved in the ubiquitin-proteasome pathway, we investigated the role of Cul-4A in regulating DDB2. Here we show that DDB2 is a specific target of Cul-4A. Coexpression of Cul-4A, but not Cul-1 or other highly related cullins, increases the ubiquitination and the decay rate of DDB2. A naturally occurring mutant of DDB2 (2RO), which does not bind Cul-4A, is not affected by coexpression of Cul-4A. Studies presented here identify a specific function of the Cul-4A gene, which is amplified and overexpressed in breast cancers.
Mol Cell Biol 2001 Oct
PMID:The xeroderma pigmentosum group E gene product DDB2 is a specific target of cullin 4A in mammalian cells. 1156 59

GCN5 is a histone acetyltransferase (HAT) originally identified in Saccharomyces cerevisiae and required for transcription of specific genes within chromatin as part of the SAGA (SPT-ADA-GCN5 acetylase) coactivator complex. Mammalian cells have two distinct GCN5 homologs (PCAF and GCN5L) that have been found in three different SAGA-like complexes (PCAF complex, TFTC [TATA-binding-protein-free TAF(II)-containing complex], and STAGA [SPT3-TAF(II)31-GCN5L acetylase]). The composition and roles of these mammalian HAT complexes are still poorly characterized. Here, we present the purification and characterization of the human STAGA complex. We show that STAGA contains homologs of most yeast SAGA components, including two novel human proteins with histone-like folds and sequence relationships to yeast SPT7 and ADA1. Furthermore, we demonstrate that STAGA has acetyl coenzyme A-dependent transcriptional coactivator functions from a chromatin-assembled template in vitro and associates in HeLa cells with spliceosome-associated protein 130 (SAP130) and DDB1, two structurally related proteins. SAP130 is a component of the splicing factor SF3b that associates with U2 snRNP and is recruited to prespliceosomal complexes. DDB1 (p127) is a UV-damaged-DNA-binding protein that is involved, as part of a complex with DDB2 (p48), in nucleotide excision repair and the hereditary disease xeroderma pigmentosum. Our results thus suggest cellular roles of STAGA in chromatin modification, transcription, and transcription-coupled processes through direct physical interactions with sequence-specific transcription activators and with components of the splicing and DNA repair machineries.
Mol Cell Biol 2001 Oct
PMID:Human STAGA complex is a chromatin-acetylating transcription coactivator that interacts with pre-mRNA splicing and DNA damage-binding factors in vivo. 1156 63

Human DNA polymerase eta (hPoleta) functions in the error-free replication of UV-damaged DNA, and mutations in hPoleta cause cancer-prone syndrome, the variant form of xeroderma pigmentosum. However, in spite of its key role in promoting replication through a variety of distorting DNA lesions, the manner by which hPoleta is targeted to the replication machinery stalled at a lesion site remains unknown. Here, we provide evidence for the physical interaction of hPoleta with proliferating cell nuclear antigen (PCNA) and show that mutations in the PCNA binding motif of hPoleta inactivate this interaction. PCNA, together with replication factor C and replication protein A, stimulates the DNA synthetic activity of hPoleta, and steady-state kinetic studies indicate that this stimulation accrues from an increase in the efficiency of nucleotide insertion resulting from a reduction in the apparent K(m) for the incoming nucleotide.
Mol Cell Biol 2001 Nov
PMID:Physical and functional interactions of human DNA polymerase eta with PCNA. 1158 3

The UV-sensitive V-H1 cell line has a T46I substitution mutation in the Walker A box in both alleles of XPD and lacks DNA helicase activity. We characterized three partial revertants that curiously display intermediate UV cytotoxicity (2- to 2.5-fold) but normal levels of UV-induced hprt mutations. In revertant RH1-26, the efficient removal of pyrimidine (6-4) pyrimidone photoproducts from both strands of hprt suggests that global-genomic nucleotide excision repair is normal, but the pattern of cyclobutane pyrimidine dimer removal suggests that transcription-coupled repair (TCR) is impaired. To explain the intermediate UV survival and lack of RNA synthesis recovery in RH1-26 after 10 J of UV/m(2), we propose a defect in repair-transcription coupling, i.e., the inability of the cells to resume or reinitiate transcription after the first TCR event within a transcript. All three revertants carry an R658H suppressor mutation, in one allele of revertants RH1-26 and RH1-53 and in both alleles of revertant RH1-3. Remarkably, the R658H mutation produces the clinical phenotype of trichothiodystrophy (TTD) in several patients who display intermediate UV sensitivity. The XPD(R658H) TTD protein, like XPD(T46I/R658H), is codominant when overexpressed in V-H1 cells and partially complements their UV sensitivity. Thus, the suppressing R658H substitution must restore helicase activity to the inactive XPD(T46I) protein. Based on current knowledge of helicase structure, the intragenic reversion mutation may partially compensate for the T46I mutation by perturbing the XPD structure in a way that counteracts the effect of this mutation. These findings have implications for understanding the differences between xeroderma pigmentosum and TTD and illustrate the value of suppressor genetics for studying helicase structure-function relationships.
Mol Cell Biol 2001 Nov
PMID:Restoration of nucleotide excision repair in a helicase-deficient XPD mutant from intragenic suppression by a trichothiodystrophy mutation. 1158 17

The xeroderma pigmentosum group D (XPD) protein is a subunit of transcription factor TFIIH with DNA helicase activity. TFIIH has two functions, in basal transcription and nucleotide excision repair. Mutations in XPD that affect DNA repair but not transcription result in the skin cancer-prone disorder, xeroderma pigmentosum (XP). If transcription is also affected, the result is the multi-system disorder trichothiodystrophy (TTD), in which there is no skin cancer predisposition, or in rare cases, XP combined with Cockayne syndrome. Up till now there have been no reports of combined clinical features of XP and TTD. We have now identified two patients with some features of both these disorders. One of these, XP189MA, a 3-year-old girl with sun sensitivity, mental and physical developmental delay, has XPD mutations not previously reported, and barely detectable levels of nucleotide excision repair. The other, XP38BR, a 28-year-old woman with sun sensitivity, pigmentation changes and skin cancers typical of XP, has a mutation that has been identified previously, but only in TTD patients with no features of XP. The level of repair of UV damage in XP38BR is substantially higher than that in other patients with the same mutation. With both patients, polarized light microscopy revealed a 'tiger-tail' appearance of the hair, and amino acid analysis of the hair shafts show levels of sulfur-containing proteins intermediate between those of normal and TTD individuals. Our findings highlight the complexities of genotype-phenotype relationships in the XPD gene.
Hum Mol Genet 2001 Oct 15
PMID:Two individuals with features of both xeroderma pigmentosum and trichothiodystrophy highlight the complexity of the clinical outcomes of mutations in the XPD gene. 1170 41

The transcription factor TFIIH is involved in both basal transcription and DNA repair. Mutations in the XPD helicase component of TFIIH can result in the diverse clinical features associated with xeroderma pigmentosum (XP) and trichothiodystrophy (TTD). It is generally believed that the multi-system abnormalities associated with TTD are the result of a subtle deficiency in basal transcription. However, to date, there has been no clear demonstration of a defect in expression of any specific gene in individuals with these syndromes. Here we show that the specific mutations in XPD that cause TTD result in reduced expression of the beta-globin genes in these individuals. Eleven TTD patients with characterized mutations in the XPD gene have the haematological features of beta-thalassaemia trait, and reduced levels of beta-globin synthesis and beta-globin mRNA. All these parameters were normal in three patients with XP. These findings provide the first evidence for reduced expression of a specific gene in TTD. They support the hypothesis that many of the clinical features of TTD result from inadequate expression of a diverse set of highly expressed genes.
Hum Mol Genet 2001 Nov 15
PMID:Mutations in the general transcription factor TFIIH result in beta-thalassaemia in individuals with trichothiodystrophy. 1173 44

What follows is a personal remembrance of how Dr. Richard Setlow influenced me as a young postdoctoral fellow at Oak Ridge National laboratory between 1963 and 1966. The narrative tries to place my "maturation" as a young, inexperienced scientist in the context of the cultural upheaval caused by the Vietnam war, of a Northerner facing a "culture-shock" living in the South and in a revolution in molecular and radiation biology taking place at Oak Ridge National Laboratory at that time. The unique historic juxtaposition of Dr. Setlow's contribution of the discovery of UV-induced pyrimidine dimers in bacterial DNA, being potentially the molecular lesion responsible for cell killing and mutagenesis, occurring as I was at Oak Ridge, and the wonderful working relationship I had with William Carrier, his technician, led to our discovery with James Regan that normal human cells repaired these lesion from their DNA. Amazingly, because of Dr. Setlow's challenge to me about my thoughts of the implications of his findings in bacteria, the chance visit to Oak Ridge National Laboratory by Dr. James Cleaver and my background as a human geneticist provided me the extraordinary opportunity to carry out a collaboration to test if human cancer prone syndromes might be deficient in the repair of these UV-induced DNA lesions. With our finding that the direct demonstration of a lack of repair of UV-induced pyrimidine dimers in cells from the skin cancer prone syndrome, xeroderma pigmentosum, opened up a new paradigm for the understanding of the molecular mechanism of carcinogenesis of both radiation and chemical carcinogenesis. From this investigator's vantage point in the history of the understanding of carcinogenesis, which has led us to the present point of "oncogenes" and "tumor suppressor genes", the old adage by Newton, "I only saw further because I stood on the shoulder of giants", is so applicable here. Dr. Setlow's shoulders were indeed among those of all of us that have made some small contribution in trying to understand this extremely complex process of human carcinogenesis.
Environ Mol Mutagen 2001
PMID:From bacteria to humans: lessons learned from a reductionist's view of ultraviolet light-induced DNA lesions. 1174 44


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