Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Originally detected in fixed cells, DNA replication foci (RFi) were later visualized in living cells by using green fluorescent protein (GFP)-tagged proliferating cell nuclear antigen (PCNA) and DNA ligase I. It was shown using fluorescence redistribution after photobleaching (FRAP) assay that focal GFP-PCNA slowly exchanged, suggesting the existence of a stable replication holocomplex. Here, we used the FRAP assay to study the dynamics of the GFP-tagged PCNA-binding proteins: Flap endonuclease 1 (Fen1) and DNA polymerase eta (Pol eta). We also used the GFP-Cockayne syndrome group A (CSA) protein, which does associate with transcription foci after DNA damage. In normal cells, GFP-Pol eta and GFP-Fen1 are mobile with residence times at RFi (t(m)) approximately 2 and approximately 0.8 s, respectively. GFP-CSA is also mobile but does not concentrate at discrete foci. After methyl methanesulfonate (MMS) damage, the mobile fraction of focal GFP-Fen1 decreased and t(m) increased, but it then recovered. The mobilities of focal GFP-Pol eta and GFP-PCNA did not change after MMS. The mobility of GFP-CSA did not change after UV-irradiation. These data indicate that the normal replication complex contains at least two mobile subunits. The decrease of the mobile fraction of focal GFP-Fen1 after DNA damage suggests that Fen1 exchange depends on the rate of movement of replication forks.
Mol Biol Cell 2005 May
PMID:High mobility of flap endonuclease 1 and DNA polymerase eta associated with replication foci in mammalian S-phase nucleus. 1575 26

Cockayne syndrome (CS) is a rare genetic disorder characterized as a segmental premature-aging syndrome. The CS group B (CSB) protein has previously been implicated in transcription-coupled repair, transcriptional elongation, and restoration of RNA synthesis after DNA damage. Recently, evidence for a role of CSB in base excision repair of oxidative DNA lesions has accumulated. In our search to understand the molecular function of CSB in this process, we identify a physical and functional interaction between CSB and poly(ADP-ribose) polymerase-1 (PARP-1). PARP-1 is a nuclear enzyme that protects the integrity of the genome by responding to oxidative DNA damage and facilitating DNA repair. PARP-1 binds to single-strand DNA breaks which activate the catalytic ability of PARP-1 to add polymers of ADP-ribose to various proteins. We find that CSB is present at sites of activated PARP-1 after oxidative stress, identify CSB as a new substrate of PARP-1, and demonstrate that poly(ADP-ribosyl)ation of CSB inhibits its DNA-dependent ATPase activity. Furthermore, we find that CSB-deficient cell lines are hypersensitive to inhibition of PARP. Our results implicate CSB in the PARP-1 poly(ADP-ribosyl)ation response after oxidative stress and thus suggest a novel role of CSB in the cellular response to oxidative damage.
Mol Cell Biol 2005 Sep
PMID:Cooperation of the Cockayne syndrome group B protein and poly(ADP-ribose) polymerase 1 in the response to oxidative stress. 1610 9

Defects in the XPD gene can result in several clinical phenotypes, including xeroderma pigmentosum (XP), trichothiodystrophy, and, less frequently, the combined phenotype of XP and Cockayne syndrome (XP-D/CS). We previously showed that in cells from two XP-D/CS patients, breaks were introduced into cellular DNA on exposure to UV damage, but these breaks were not at the sites of the damage. In the present work, we show that three further XP-D/CS patients show the same peculiar breakage phenomenon. We show that these breaks can be visualized inside the cells by immunofluorescence using antibodies to either gamma-H2AX or poly-ADP-ribose and that they can be generated by the introduction of plasmids harboring methylation or oxidative damage as well as by UV photoproducts. Inhibition of RNA polymerase II transcription by four different inhibitors dramatically reduced the number of UV-induced breaks. Furthermore, the breaks were dependent on the nucleotide excision repair (NER) machinery. These data are consistent with our hypothesis that the NER machinery introduces the breaks at sites of transcription initiation. During transcription in UV-irradiated XP-D/CS cells, phosphorylation of the carboxy-terminal domain of RNA polymerase II occurred normally, but the elongating form of the polymerase remained blocked at lesions and was eventually degraded.
Mol Cell Biol 2005 Sep
PMID:Transcription-associated breaks in xeroderma pigmentosum group D cells from patients with combined features of xeroderma pigmentosum and Cockayne syndrome. 1613 23

Loss of a nonenzymatic function of XPG results in defective transcription-coupled repair (TCR), Cockayne syndrome (CS), and early death, but the molecular basis for these phenotypes is unknown. Mutation of CSB, CSA, or the TFIIH helicases XPB and XPD can also cause defective TCR and CS. We show that XPG interacts with elongating RNA polymerase II (RNAPII) in the cell and binds stalled RNAPII ternary complexes in vitro both independently and cooperatively with CSB. XPG binds transcription-sized DNA bubbles through two domains not required for incision and functionally interacts with CSB on these bubbles to stimulate its ATPase activity. Bound RNAPII blocks bubble incision by XPG, but an ATP hydrolysis-dependent process involving TFIIH creates access to the junction, allowing incision. Together, these results implicate coordinated recognition of stalled transcription by XPG and CSB in TCR initiation and suggest that TFIIH-dependent remodeling of stalled RNAPII without release may be sufficient to allow repair.
Mol Cell 2005 Oct 28
PMID:Recognition of RNA polymerase II and transcription bubbles by XPG, CSB, and TFIIH: insights for transcription-coupled repair and Cockayne Syndrome. 1624 22

Although genotoxic agents are powerful inducers of stress kinases (SAPK/JNK), the contribution of DNA damage itself to this response is unknown. Therefore, SAPK/JNK activation of cells harboring specific defects in DNA damage-recognition mechanisms was studied. Dual phosphorylation of SAPK/JNK by the genotoxin methyl methanesulfonate (MMS) occurred in two waves. The early response (< or = 2 h after exposure) was similar in cells knockout for ATM, PARP, p53, and CSB or defective in DNA-PK(cs) compared with wild-type cells. The late response however (> or = 4 h), was drastically reduced in DNA-PK(cs) and Cockayne's syndrome B (CSB)-deficient cells. Similar results were obtained with human cells lacking DNA-PK(cs) and CSB. Activation of SAPK/JNK by MMS was not affected upon inhibition of base excision repair (BER), indicating base damage itself does not signal to SAPK/JNK. Because SAPK/JNK activation was attenuated in nongrowing cells, DNA replication-dependent processing of lesions, involving DNA-PK(cs) and CSB, appears to be required. DNA-PK(cs) coprecipitates with SEK1/MKK4 and SAPK/JNK, supporting a role of DNA-PK(cs) in SAPK/JNK activation. In this process, Rho GTPases are involved since inhibition of Rho impairs MMS-induced signaling to SAPK/JNK. The data show that sensing of DNA damage by DNA-PK(cs) and CSB causes a delayed SEK1/MKK4-mediated dual phosphorylation of SAPK/JNK.
Mol Biol Cell 2006 Feb
PMID:Late activation of stress kinases (SAPK/JNK) by genotoxins requires the DNA repair proteins DNA-PKcs and CSB. 1631 74

Genetic alterations affecting nucleotide excision repair, the most versatile DNA-repair mechanism responsible for removal of bulky DNA adducts including ultraviolet (UV) light-induced DNA lesions, may result in the rare, recessively inherited autosomal syndromes xeroderma pigmentosum (XP), Cockayne syndrome (CS), or trichothiodystrophy (TTD). Classical approaches such as somatic cell fusions or microinjection assays have formalized the genetic complexity of these related but clinically distinct syndromes, and contributed to the determination of seven, five, and three complementation groups for XP, CS, and TTD, respectively. XP patients are highly susceptible to photoinduced cutaneous cancers of epidermal origin. To better study the responses to UV irradiation of XP keratinocytes, and to objectively determine the extent to which cutaneous gene therapy may be realized, we set up experimental procedures adapted to ex vivo genetic complementation of keratinocytes from XP patients. We provide here detailed rationales and procedures for these approaches.
Methods Mol Biol 2006
PMID:Complementation assays adapted for DNA repair-deficient keratinocytes. 1667 70

Nucleotide excision repair (NER) is the most versatile and best studied DNA repair system in humans. NER can repair a variety of bulky DNA damages including UV-light induced DNA photoproducts. NER consists of a multistep process in which the DNA lesion is recognized and demarcated by DNA unwinding. Then, an approximately 28 bp DNA damage containing oligonucleotide is excised followed by gap filling using the undamaged DNA strand as a template. The consequences of defective NER are demonstrated by three rare autosomal-recessive NER-defective syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS), and trichothiodystrophy (TTD). XP patients show severe sun sensitivity, freckling in sun exposed skin, and develop skin cancers already during childhood. CS patients exhibit sun sensitivity, severe neurologic abnormalities, and cachectic dwarfism. Clinical symptoms of TTD patients include sun sensitivity, freckling in sun exposed skin areas, and brittle sulfur-deficient hair. In contrast to XP patients, CS and TTD patients are not skin cancer prone. Studying these syndromes can increase the knowledge of skin cancer development including cutaneous melanoma as well as basal and squamous cell carcinoma in general that may lead to new preventional and therapeutic anticancer strategies in the normal population.
J Mol Histol 2006 Sep
PMID:Nucleotide excision repair and cancer. 1685 87

Rpb9, a small nonessential subunit of RNA polymerase II, has been shown to have multiple transcription-related functions in Saccharomyces cerevisiae. These functions include promoting transcription elongation and mediating a subpathway of transcription-coupled repair (TCR) that is independent of Rad26, the homologue of human Cockayne syndrome complementation group B protein. Rpb9 is composed of three distinct domains: the N-terminal Zn1, the C-terminal Zn2, and the central linker. Here we show that the Zn1 and linker domains are essential, whereas the Zn2 domain is almost dispensable, for both transcription elongation and TCR functions. Impairment of transcription elongation, which does not dramatically compromise Rad26-mediated TCR, completely abolishes Rpb9-mediated TCR. Furthermore, Rpb9 appears to be dispensable for TCR if its transcription elongation function is compensated for by removing a transcription repression/elongation factor. Our data suggest that the transcription elongation function of Rpb9 is involved in TCR.
Mol Cell Biol 2006 Dec
PMID:Evidence that the transcription elongation function of Rpb9 is involved in transcription-coupled DNA repair in Saccharomyces cerevisiae. 1703 Jun 4

Mutations in the CSB gene cause Cockayne syndrome (CS), a DNA repair disorder characterized by UV sensitivity and severe physical and neurological impairment. CSB functions in the transcription-coupled repair subpathway of nucleotide excision repair. This function may explain the UV sensitivity but hardly clarifies the other CS symptoms. Many of these, including retinopathy, are associated with premature aging. We studied eye pathology in a mouse model for CS. Csb(m/m) mice were hypersensitive to UV light and developed epithelial hyperplasia and squamous cell carcinomas in the cornea, which underscores the importance of transcription-coupled repair of photolesions in the mouse. In addition, we observed a spontaneous loss of retinal photoreceptor cells with age in the Csb(m/m) retina, resulting in a 60% decrease in the number of rods by the age of 18 months. Importantly, when Csb(m/m) mice (as well as Csa(-/-) mice) were exposed to 10 Gy of ionizing radiation, we noticed an increase in apoptotic photoreceptor cells, which was not observed in wild-type animals. This finding, together with our observation that the expression of established oxidative stress marker genes is upregulated in the Csb(m/m) retina, suggests that (endogenous) oxidative DNA lesions play a role in this CS-specific premature-aging feature and supports the oxidative DNA damage theory of aging.
Mol Cell Biol 2007 Feb
PMID:Retinal degeneration and ionizing radiation hypersensitivity in a mouse model for Cockayne syndrome. 1714 77

Transcription-coupled repair (TCR) efficiently removes a variety of lesions from the transcribed strand of active genes. Mutations in Cockayne syndrome group A and B genes (CSA and CSB) result in defective TCR, but the molecular mechanism of TCR in mammalian cells is not clear. We have found that CSA protein is translocated to the nuclear matrix after UV irradiation and colocalized with the hyperphosphorylated form of RNA polymerase II and that the translocation is dependent on CSB. We developed a cell-free system for the UV-induced translocation of CSA. A cytoskeleton (CSK) buffer-soluble fraction containing CSA and a CSK buffer-insoluble fraction prepared from UV-irradiated CS-A cells were mixed. After incubation, the insoluble fraction was treated with DNase I. CSA protein was detected in the DNase I-insoluble fraction, indicating that it was translocated to the nuclear matrix. In this cell-free system, the translocation was dependent on UV irradiation, CSB function, and TCR-competent CSA. Moreover, the translocation was dependent on functional TFIIH, as well as chromatin structure and transcription elongation. These results suggest that alterations of chromatin at the RNA polymerase II stall site, which depend on CSB and TFIIH at least, are necessary for the UV-induced translocation of CSA to the nuclear matrix.
Mol Cell Biol 2007 Apr
PMID:Functional TFIIH is required for UV-induced translocation of CSA to the nuclear matrix. 1724 93


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