Biomedical subjects

J M Egly

Publications and source records attributed to J M Egly.

At least 19 recordsLinked to original sources

PubMed1995

The transcriptional elongation inhibitor 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole inhibits transcription factor IIH-associated protein kinase.

Regulation of chain elongation by RNA polymerase II can have an important effect on gene expression (Bentley, D. (1995) Curr. Opin. Genet. Dev. 5, 210-216; Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759); however the mechanisms that control this step in transcription are not well understood. The adenosine analogue 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) has long been used as an inhibitor of RNA polymerase II elongation, but its target is not known. We show that DRB is a potent inhibitor of Cdk-activating kinase, associated with the general transcription factor TFIIH. Two other inhibitors of this kinase, H-7 and H-8, also inhibited transcriptional elongation. Furthermore, TFIIH kinase bound specifically to the herpes simplex virus VP16 activation domain which stimulates polymerase II elongation in addition to initiation (Yankulov, K., Blau, J., Purton, T., Roberts, S., and Bentley, D. (1994) Cell 77, 749-759). Our results suggest that DRB affects transcription by inhibiting the TFIIH-associated kinase and that this kinase functions in the control of elongation by RNA polymerase II.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed1995

Genomic structure of the human TATA-box-binding protein (TBP).

The gene encoding the human TATA-box-binding protein (hTBP) is contained within a 20-kb DNA fragment and is split into eight exons. The coding sequence is interrupted by six introns and the 5'-untranslated region (5'-UTR) of the gene by a 2.5-kb intron. A comparison of the hTBP exon/intron organization with the various TBP cloned to date is presented.

Amino Acid Sequence↗

PubMed1995

AUC is used as a start codon in Escherichia coli.

Overexpression of the polymerase II transcription factor E subunit alpha mRNA (TFIIE alpha) in Escherichia coli with the pET3a expression vector [Studier et al., Methods Enzymol. 185 (1990) 60-89] results in the production of a 50-kDa N-truncated polypeptide in addition to the 57-kDa wild-type polypeptide. Microsequencing analysis of the 50-kDa truncated protein and verification of the TFIIE alpha mRNA, showed that a Shine-Dalgarno-like sequence present 200 nucleotides downstream from the normal start codon could direct the use of an internal AUC codon. This codon should thus be added to the list of the non-AUG start codons.

Amino Acid Sequence↗

PubMed1995

Mammalian DNA nucleotide excision repair reconstituted with purified protein components.

Nucleotide excision repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete repair synthesis was achieved by combining these factors with DNA polymerase epsilon, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.

Animals↗

PubMed1995

Partial characterization of the DNA repair protein complex, containing the ERCC1, ERCC4, ERCC11 and XPF correcting activities.

The nucleotide excision repair (NER) protein ERCC1 is part of a functional complex, which harbors in addition the repair correcting activities of ERCC4, ERCC11 and human XPF. ERCC1 is not associated with a defect in any of the known human NER disorders: xeroderma pigmentosum, Cockayne's syndrome or trichothiodystrophy. Here we report the partial purification and characterization of the ERCC1 complex. Immunoprecipitation studies tentatively identified a subunit in the complex with an apparent MW of approximately 120 kDa. The complex has affinity for DNA, but no clear preference for ss, ds or UV-damaged DNA substrates. The size of the entire complex determined by non-denaturing gradient gels (approximately 280 kDa) is considerably larger than previously found using size separation on glycerol gradients (approximately 120 kDa). Stable associations of the ERCC1 complex with other known repair factors (XPA, XPC, XPG and TFIIH complex) could not be detected.

Animals↗

PubMed1995

TFIIH: a link between transcription, DNA repair and cell cycle regulation.

TFIIH is a basal transcription factor for protein-coding genes. It contains ERCC2, ERCC3, MO15 and cyclin H, polypeptides implicated in nucleotide excision repair or cell cycle regulation. The dysfunction of TFIIH could result in a large panel of genetic disorders, such as xeroderma pigmentosum, Cockayne's syndrome and trichothiodystrophy. This link between transcription, DNA repair and cell cycle has highlighted a complex and essential role for TFIIH in the cell and has provided much information on the molecular mechanisms of each of these cellular processes.

Cell Cycle↗

PubMed1995

p53 modulation of TFIIH-associated nucleotide excision repair activity.

p53 has pleiotropic functions including control of genomic plasticity and integrity. Here we report that p53 can bind to several transcription factor IIH-associated factors, including transcription-repair factors, XPD (Rad3) and XPB, as well as CSB involved in strand-specific DNA repair, via its C-terminal domain. We also found that wild-type, but not Arg273His mutant p53 inhibits XPD (Rad3) and XPB DNA helicase activities. Moreover, repair of UV-induced dimers is slower in Li-Fraumeni syndrome cells (heterozygote p53 mutant) than in normal human cells. Our findings indicate that p53 may play a direct role in modulating nucleotide excision repair pathways.

Adenosine Triphosphatases↗

PubMed1994

The MO15 cell cycle kinase is associated with the TFIIH transcription-DNA repair factor.

A protein kinase activity that phosphorylates the C-terminal domain (CTD) of RNA polymerase II and is associated with the basal transcription-repair factor TFIIH (also called BTF2) resides with MO15, a cyclin-dependent protein kinase that was first found to be involved in cell cycle regulation. Using in vivo and in vitro repair assays, we show that MO15 is important for nucleotide excision repair, most likely through its association with TFIIH, thus providing an unexpected link among three important cellular mechanisms.

Amino Acid Sequence↗

PubMed1994

The ERCC2/DNA repair protein is associated with the class II BTF2/TFIIH transcription factor.

ERCC2 is involved in the DNA repair syndrome xeroderma pigmentosum (XP) group D and was found to copurify with the RNA polymerase II (B) transcription factor BTF2/TFIIH that possesses a bidirectional helicase activity. Antibodies directed towards the 89 kDa (ERCC3) or the p62 subunit of BTF2 are able to either immunoprecipitate ERCC2 or shift the polypeptide in a glycerol gradient. Conversely, an antibody directed towards ERCC2 also retains or shifts BTF2. ERCC2 could be resolved from the other characterized components of BTF2 upon salt treatment, while its readdition enhanced BTF2 transcription activity. ERCC2, ERCC3 and p44 are three repair proteins found in association with BTF2. Two of them, ERCC2 and ERCC3, are responsible for atypical forms of XP disorders which confer a high predisposition to skin cancer. This includes clinical features that lack an adequate rationalization on the basis of nucleotide excision repair (NER) deficiency but which may now be explained better in terms of a partial transcription deficiency.

Amino Acid Sequence↗

PubMed1994

p44 and p34 subunits of the BTF2/TFIIH transcription factor have homologies with SSL1, a yeast protein involved in DNA repair.

The human BTF2 (TFIIH) transcription factor is a multisubunit protein involved in transcription initiation by RNA polymerase II (B) as well as in DNA repair. In addition to the previously characterized p62 and p89/ERCC3 subunits, we have cloned two other subunits of BTF2, p44 and p34. The gene encoding p44 appeared to be the human counterpart of SSL1, a gene involved in translation and UV resistance in yeast. Interestingly, the p34 subunit also has homology with a domain of SSL1, suggesting that it corresponds to an as yet unidentified protein involved in DNA repair. Both p44 and p34 possess zinc finger domains that may mediate BTF2 binding to nucleic acids.

Amino Acid Sequence↗

PubMed1994

The DNA-dependent ATPase activity associated with the class II basic transcription factor BTF2/TFIIH.

BTF2/TFIIH from human, delta from rat, and factor b from yeast are multisubunit basal transcription factors that have been shown to be closely associated with a protein kinase capable of phosphorylating the carboxyl-terminal domain of the large subunit of RNA polymerase II (Lu, H., Zawel, L., Fischer, L., Egly, J. M., and Reinberg, D. (1992) Nature 358, 641-645; Serizawa, H., Conaway, R. C., and Conaway, J. W. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7476-7480; Feaver, W. J., Gileadi, O., and Kornberg, R. D. (1991) Cell 67, 1223-1230). We report here that a DNA-dependent ATPase and the previously characterized helicase (Schaeffer, L., Roy, R., Humbert, S., Moncollin, V., Vermeulen, W., Hoeijmakers, J., Chambon, P., and Egly, J. M. (1993) Science 260, 58-63) are both associated with BTF2 and reside with the p89 polypeptide subunit. The DNA requirement, the effect of Sarkosyl and staurosporine inhibitors, as well as nucleotide competition experiments, clearly distinguished ATPase/helicase from the carboxyl-terminal domain kinase. Using recombinant wild type or mutated p89/ERCC3 polypeptides and different forms of DNA template, we show the connection between ATPase and the helicase.

Adenosine Triphosphatases↗

PubMed1994

Transcription by RNA polymerase II: a process linked to DNA repair.

The proteins that are implicated in the basal transcription of protein coding genes have now been identified. Although little is known about their function, recent data demonstrate the ability of these proteins, previously called class II transcription factors, to participate in other reactions: TBP, the TATA-box binding factor, is involved in class I and III transcription, while TFIIH has been shown to possess components that are involved in the DNA repair mechanism. The involvement of some if not all of the TFIIH subunits in transcription and repair may explain the heterogeneity of the various and sometimes completely unrelated symptoms observed in xeroderma pigmentosum, Cockayne Syndrome and trichothiodystrophy disorders.

Animals↗

PubMed1994

Expression in Escherichia coli: production and purification of both subunits of the human general transcription factor TFIIE.

Both subunits of the human class II transcription factor TFIIE (rTFIIE alpha and rTFIIE beta) have been overexpressed in Escherichia coli at 26 degrees C using a T7 RNA polymerase expression system and further purified to apparent homogeneity. As in this system rTFIIE alpha was poorly expressed and copurified with a truncated form, we expressed rTFIIE alpha as a fusion protein. These overexpressed subunits of TFIIE are similar to the endogenous subunits according to the following criteria: molecular weight, microsequencing, and transcription activity.

Affinity Labels↗

PubMed1994

Ion exchange resins for the purification of monoclonal antibodies from animal cell culture.

We have compared various ion exchangers for monoclonal antibody (MAb) purification using different starting materials such as ascitic fluid and cell culture supernatant. Twelve cation and anion exchange resins were tested so far. Purification of MAbs with regard to the starting material is described. In well-defined conditions of adsorption (20 mM MES buffer, pH 6.50), one purification step based on cation-exchange chromatography is generally sufficient to achieve at least 90% purity of the MAb, even when produced by animal cell culture. Cation-exchange supports exhibit higher capacity for MAbs compared to anion exchangers. Among the cation exchangers tested, we have selected the cross-linked matrix S Sepharose FF for its large specificity and capacity for MAbs. Considering these key parameters and also the good mechanical resistance of the S Sepharose FF, we describe how, by varying the flow rate, sample concentration, and size of the column, the productivity may be improved in a monoclonal antibody purification process. Finally, a general 'gram scale' purification protocol of MAbs produced by animal cell cultures is proposed. This protocol, based on economical adsorption conditions and three steps of elution (100 mM, 200 mM and 1 M NaCl), allows the recovery of highly purified MAbs.

Animals↗

PubMed1993

The major histocompatibility complex (MHC) Ea promoter: sequences and factors at the initiation site.

We have analysed the function of sequences in the TATA/initiator region of the promoter of Ea, a class II gene of the Major Histocompatibility Complex. We find that the Ea promoter contains an initiator element with a strong influence on transcription. We also find that the Ea promoter does contain a bona fide TATA box, which can be recognized by the TATA binding protein (TBP), and that TBP is required for transcriptional activity. For activity, TBP must be included within a larger TFIID complex, as Ea transcription in a heat-treated extract can be restored by immunopurified TFIID but not by TBP alone. On the other hand, the TATA motif can be eliminated without significantly affecting either the efficiency or the startsites of transcription. This suggests that TBP, even in this TATA-containing promoter, is held in place by other components of the initiation complex, regardless of its affinity for the underlying DNA.

Animals↗

PubMed1993

A TBP-containing multiprotein complex (TIF-IB) mediates transcription specificity of murine RNA polymerase I.

TIF-IB is a transcription factor which interacts with the mouse ribosomal gene promoter and nucleates the formation of an initiation complex containing RNA polymerase I (Pol I). We have purified this factor to near homogeneity and demonstrate that TIF-IB is a large complex (< 200 kDa) which contains several polypeptides. One of the subunits present in this protein complex is the TATA-binding protein (TBP) as revealed by copurification of TIF-IB activity and TBP over different chromatographic steps including immunoaffinity purification. In addition to TBP, three tightly associated proteins (TAFs-I) with apparent molecular weights of 95, 68, and 48 kDa are contained in this multimeric complex. This subunit composition is similar--but not identical--to the analogous human factor SL1. Depletion of TBP from TIF-IB-containing fractions by immunoprecipitation eliminates TIF-IB activity. Neither TBP alone nor fractions containing other TBP complexes are capable of substituting for TIF-IB activity. Therefore, TIF-IB is a unique complex with Pol I-specific TAFs distinct from other TBP-containing complexes. The identification of TBP as an integral part of the murine rDNA promoter-specific transcription initiation factor extends the previously noted similarity of transcriptional initiation by the three nuclear RNA polymerases and underscores the importance of TAFs in determining promoter specificity.

Animals↗

PubMed1993

Common and unique transcription factor requirements of human U1 and U6 snRNA genes.

The human U1 and U6 genes have similar basal promoter structures. A first analysis of the factor requirements for the transcription of a human U1 gene by RNA polymerase II in vitro has been undertaken, and these requirements compared with those of human U6 gene transcription by RNA polymerase III in the same extracts. Fractions containing PSE-binding protein (PBP) are shown to be essential for transcription of both genes, and further evidence that PBP itself is required for U1 as well as U6 transcription is presented. On the other hand, the two genes have distinct requirements for TATA-binding protein (TBP). On the basis of chromatographic and functional properties, the TBP, or TBP complex, required for U1 transcription appears to differ from previously described complexes required for RNA polymerase I, II or III transcription. The different TBP requirements of the U1 and U6 promoters are reflected by specific association with either TFIIB or TFIIIB respectively, thus providing a basis for differential RNA polymerase selection.

Base Sequence↗

PubMed1993

Functional and biochemical interaction of the HTLV-I Tax1 transactivator with TBP.

The human T-cell leukemia virus type I (HTLV-I) codes for the potent transcriptional activator, Tax1, which induces the enhancer activity of various enhancer elements. In the case of the 21 bp enhancer of the HTLV-I provirus, this induction is correlated with the association of Tax1 with this DNA element via a specific cellular factor. That the indirect association of Tax1 with DNA can lead to transcriptional activation has also been supported by the study of chimeric GAL4-Tax1 proteins. The GAL4-Tax1 stimulatory effect exhibits a strong self-squelching. In order to determine whether Tax1 interacts directly with the general transcription factors or via intermediary molecules, we have analyzed how overexpression of the TATA binding protein (TBP) and TFIIB protein affects the squelching curve of GAL4-Tax1. The data presented here show that overexpression of TBP strongly increases the stimulatory effect of GAL4-Tax1, causes a displacement of the maximum of the squelching curve and partially alleviates the squelching. Under similar conditions TFIIB exhibited little effect. From these results we conclude that Tax1 can increase the recruitment of TBP by directly interacting with this protein. Biochemical experiments with purified proteins produced in bacteria confirmed that Tax1 can interact with TBP but not with TFIIB. Tax1 interacts with the conserved C-terminal part of TBP. Analysis of the ability of different mutants of Tax1 fused to the GAL4 DNA binding domain to activate transcription and to associate with TBP, showed that these activities are correlated. However, since one transcriptionally inactive mutant was able to interact efficiently with TBP in vitro, it would appear that an event other than the Tax1-TBP contact also intervenes in the activation of transcription by Tax1.

DNA-Binding Proteins↗