Biomedical subjects

V Moncollin

Publications and source records attributed to V Moncollin.

At least 19 recordsLinked to original sources

PubMed1999

Cyclin-dependent kinase and Cks/Suc1 interact with the proteasome in yeast to control proteolysis of M-phase targets.

Cell cycle-specific proteolysis is critical for proper execution of mitosis in all eukaryotes. Ubiquitination and subsequent proteolysis of the mitotic regulators Clb2 and Pds1 depend on the cyclosome/APC and the 26S proteasome. We report here that components of the cell cycle machinery in yeast, specifically the cell cycle regulatory cyclin-dependent kinase Cdc28 and a conserved associated protein Cks1/Suc1, interact genetically, physically, and functionally with components of the 26S proteasome. A mutation in Cdc28 (cdc28-1N) that interferes with Cks1 binding, or inactivation of Cks1 itself, confers stabilization of Clb2, the principal mitotic B-type cyclin in budding yeast. Surprisingly, Clb2-ubiquitination in vivo and in vitro is not affected by mutations in cks1, indicating that Cks1 is not essential for cyclosome/APC activity. However, mutant Cks1 proteins no longer physically interact with the proteasome, suggesting that Cks1 is required for some aspect of proteasome function during M-phase-specific proteolysis. We further provide evidence that Cks1 function is required for degradation of the anaphase inhibitor Pds1. Stabilization of Pds1 is partially responsible for the metaphase arrest phenotype of cks1 mutants because deletion of PDS1 partially relieves the metaphase block in these mutants.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Molecular cloning of a rat 49-kDa TBP-interacting protein (TIP49) that is highly homologous to the bacterial RuvB.

TBP as a central component in transcriptional regulation can form complexes with various regulatory factors. Using histidine-tagged TBP for affinity-purification of TBP-bound proteins, we isolated a 49-kD protein termed TBP-interacting protein 49 (TIP49) from rat liver nuclear extracts. We cloned the entire cDNA of TIP49 encoding a novel polypeptide of 456 amino acids, and thereafter established an FM3A cell line that constitutively expressed an epitope-tagged TBP. Immunoprecipitation analysis of the cell extracts indicated that TIP49 and TBP were present in an identical complex. Interestingly, the amino acid sequence of TIP49 exhibited high similarity to those sequences of the RuvB bacterial recombination factors which direct branch migration of the Holliday junction and contain the Walker A and B motifs responsible for ATP binding and ATP hydrolysis. These findings suggest that TIP49 is a putative ATP-dependent DNA helicase.

ATPases Associated with Diverse Cellular Activitie↗

PubMed1996

A 3' --> 5' XPB helicase defect in repair/transcription factor TFIIH of xeroderma pigmentosum group B affects both DNA repair and transcription.

XPB is a subunit of the basal transcription factor TFIIH, which is also involved in nucleotide excision repair (NER) and potentially in cell cycle regulation. A frameshift mutation in the 3'-end of the XPB gene is responsible for a concurrence of two disorders: xeroderma pigmentosum (XP) and Cockayne's syndrome (CS). We have isolated TFIIH from cells derived from a patient (XP11BE) who carries this frameshift mutation (TFIIHmut) and from the mother of this patient (TFIIHwt) to determine the biochemical consequences of the mutation. Although identical in composition and stoichiometry to TFIIHwt, TFIIHmut shows a reduced 3' --> 5' XPB helicase activity. A decrease in helicase and DNA-dependent ATPase activities was also observed with the mutated recombinant XPB protein. The XPB mutation causes a severe NER defect. In addition, we provide evidence for a decrease in basal transcription activity in vitro. The latter defect may provide an explanation for many of the XP and CS symptoms that are difficult to rationalize based solely on an NER defect. Thus, this work presents the first detailed analysis of a naturally occurring mutation in a basal transcription factor and supports the concept that the combined XP/CS clinical entity is actually the result of a combined transcription/repair deficiency.

Amino Acid Sequence↗

PubMed1996

MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH.

MAT1, cyclin H and cdk7 are part of TFIIH, a class II transcription factor which possesses numerous subunits of which several have been shown to be involved in processes other than transcription. Two of them, XPD (ERCC2) and XPB (ERCC3), are helicases involved in nucleotide excision repair (NER), whereas cdk7, cyclin H and MAT1 are thought to participate in cell cycle regulation. MAT1, cyclin H and cdk7 exist as a ternary complex either free or associated with TFIIH from which the latter can be dissociated at high salt concentration. MAT1 is strongly associated with cdk7 and cyclin H. Although not strictly required for the formation and activity of the complex, it stimulates its kinase activity. The kinase activity of TFIIH, which is constant during the cell cycle, is reduced after UV light irradiation.

Amino Acid Sequence↗

PubMed1996

Structures of the rat proteasomal ATPases: determination of highly conserved structural motifs and rules for their spacing.

We have isolated rat cDNAs for all of the five known proteasomal ATPases. The protein sequences of rat TBP1, TBP7, MSS1, S4, and SUG1 predicted from the open reading frames consist of 439, 418, 433, 440, and 406 amino acid residues, respectively, and exhibit striking similarities to each human counterpart with only several amino acid substitutions. These five rat ATPases are also highly homologous with each other. The N-terminal region in rat TBP1, TBP7, and SUG1 contains a heptad repeat of hydrophobic amino acids reminiscent of a leucine zipper. Also, in the central region of each rat ATPase, we found four conserved motifs, Gx4GKT, DEID, SAT, and H/QRxGRx2R, that are characteristic of a large family of ATP-dependent RNA/DNA helicases. The spacing between individual motifs was strictly conserved in the rat ATPases. These findings suggest a common function of the rat proteasomal ATPases in ATP-dependent RNA/DNA unwinding.

ATPases Associated with Diverse Cellular Activitie↗

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

Distamycin A and tallimustine inhibit TBP binding and basal in vitro transcription.

The antibiotic distamycin A is a DNA minor groove binding drug (MGB) that recognizes a stretch of at least four ATs. The alkylating benzoyl mustard derivative tallimustine (FCE 24517) has powerful anti-tumor activity. Using the electrophoretic mobility shift assay (EMSA) we determined that both compounds can prevent binding of TBP and, with 10-fold higher concentration, TBP-TFIIA (DA) and TBP-TFIIA-TFIIB (DAB) to a TATA box. Once formed, the DA and DAB complexes are more resistant to MGB challenge. Both drugs can inhibit basal in vitro transcription of a minimal TATA-containing promoter and similar concentrations are necessary for binding and transcriptional inhibition. Tallimustine shows strong selectivity by decreasing only correctly initiated transcripts. Even at high doses (20 microM), however, they cannot disturb a competent pre-initiation complex or Pol II progression. This functional in vitro model will provide a way to investigate the activity of sequence-specific DNA binding drugs with potential anti-viral and anti-tumour activity and to develop novel more selective compounds.

Antineoplastic Agents↗

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 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

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↗

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

DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor.

The human BTF2 basic transcription factor (also called TFIIH), which is similar to the delta factor in rat and factor b in yeast, is required for class II gene transcription. A strand displacement assay was used to show that highly purified preparation of BTF2 had an adenosine triphosphate-dependent DNA helicase activity, in addition to the previously characterized carboxyl-terminal domain kinase activity. Amino acid sequence analysis of the tryptic digest generated from the 89-kilodalton subunit of BTF2 indicated that this polypeptide corresponded to the ERCC-3 gene product, a presumed helicase implicated in the human DNA excision repair disorders xeroderma pigmentosum and Cockayne's syndrome. These findings suggest that transcription and nucleotide excision repair may share common factors and hence may be considered to be functionally related.

Adenosine Triphosphate↗

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↗