DNA repair complex licenses acetylation of H2A.Z.1 by KAT2A during transcription.

Post-translational modifications of histone variant H2A.Z accompany gene transactivation, but its modifying enzymes still remain elusive. Here, we reveal a hitherto unknown function of human KAT2A (GCN5) as a histone acetyltransferase (HAT) of H2A.Z at the promoters of a set of transactivated genes. Expression of these genes also depends on the DNA repair complex XPC-RAD23-CEN2. We established that XPC-RAD23-CEN2 interacts both with H2A.Z and KAT2A to drive the recruitment of the HAT at promoters and license H2A.Z acetylation. KAT2A selectively acetylates H2A.Z.1 versus H2A.Z.2 in vitro on several well-defined lysines and we unveiled that alanine-14 in H2A.Z.2 is responsible for inhibiting the activity of KAT2A. Notably, the use of a nonacetylable H2A.Z.1 mutant shows that H2A.Z.1ac recruits the epigenetic reader BRD2 to promote RNA polymerase II recruitment. Our studies identify KAT2A as an H2A.Z.1 HAT in mammals and implicate XPC-RAD23-CEN2 as a transcriptional co-activator licensing the reshaping of the promoter epigenetic landscape.

XPC is an RNA polymerase II cofactor recruiting ATAC to promoters by interacting with E2F1.

The DNA damage sensor XPC is involved in nucleotide excision repair. Here we show that in the absence of damage, XPC co-localizes with RNA polymerase II (Pol II) and active post-translational histone modifications marks on a subset of class II promoters in human fibroblasts. XPC depletion triggers specific gene down-expression due to a drop in the deposition of histone H3K9 acetylation mark and pre-initiation complex formation. XPC interacts with the histone acetyltransferase KAT2A and specifically triggers the recruitment of the KAT2A-containing ATAC complex to the promoters of down-expressed genes. We show that a strong E2F1 signature characterizes the XPC/KAT2A-bound promoters and that XPC interacts with E2F1 and promotes its binding to its DNA element. Our data reveal that the DNA repair factor XPC is also an RNA polymerase II cofactor recruiting the ATAC coactivator complex to promoters by interacting with the DNA binding transcription factor E2F1.

TFIIH functions are altered by the P210BCR-ABL oncoprotein produced on the Philadelphia chromosome.

P210BCR-ABL counteracted against the complementary effect of XPB on DNA repair when ultraviolet (UV)-sensitive 27-1 cells were treated with UV or cisplatin but not with hydrogen peroxide. Wortmannin, an inhibitor of PI3 kinase did not affect its anti-repair effect. Enhanced recruitment of p44 with TFIIH after cisplatin treatment is inhibited by the expression of P210BCR-ABL in a kinase activity-dependent manner. Although purified TFIIH from P210BCR-ABL expressor and non-expressor showed almost no difference in molar ratio of each component, the in vitro activity of TFIIH was decreased by 5-10% in repair assay but was increased by more than two-fold in transcription assay.

Codominance associated with overexpression of certain XPD mutations.

Mutations in the XPD gene are associated with three complex clinical phenotypes, namely xeroderma pigmentosum (XP), XP in combination with Cockayne syndrome (XP-CS), and trichothiodystrophy (TTD). XP is caused by a deficiency in nucleotide excision repair (NER) that results in a high risk of skin cancer. TTD is characterized by severe developmental and neurological defects, with hallmark features of brittle hair and scaly skin, and sometimes has defective NER. We used CHO cells as a system to study how specific mutations alter the dominant/recessive behavior of XPD protein. Previously we identified the T46I and R75W mutations in two highly UV-sensitive hamster cell lines that were reported to have paradoxically high levels of unscheduled DNA synthesis. Here we report that these mutants have greatly reduced XPD helicase activity and fully defective NER in a cell-extract excision assay. We conclude that the unscheduled DNA synthesis seen in these mutants is caused by abortive "repair" that does not contribute to cell survival. These mutations, as well as the K48R canonical helicase-domain mutation, each produced codominant negative phenotypes when overexpressed in wild-type CHO cells. The common XP-specific R683W mutation also behaved in a codominant manner when overexpressed, which is consistent with the idea that this mutation may affect primarily the enzymatic activity of the protein rather than impairing protein interactions, which may underlie TTD. A C-terminal mutation uniquely found in TTD (R722W) was overexpressed but not to levels sufficiently high to rigorously test for a codominant phenotype. Overexpression of mutant XPD alleles may provide a simple means of producing NER deficiency in other cell lines.

Mechanism of promoter melting by the xeroderma pigmentosum complementation group B helicase of transcription factor IIH revealed by protein-DNA photo-cross-linking.

The p89/xeroderma pigmentosum complementation group B (XPB) ATPase-helicase of transcription factor IIH (TFIIH) is essential for promoter melting prior to transcription initiation by RNA polymerase II (RNAPII). By studying the topological organization of the initiation complex using site-specific protein-DNA photo-cross-linking, we have shown that p89/XPB makes promoter contacts both upstream and downstream of the initiation site. The upstream contact, which is in the region where promoter melting occurs (positions -9 to +2), requires tight DNA wrapping around RNAPII. The addition of hydrolyzable ATP tethers the template strand at positions -5 and +1 to RNAPII subunits. A mutation in p89/XPB found in a xeroderma pigmentosum patient impairs the ability of TFIIH to associate correctly with the complex and thereby melt promoter DNA. A model for open complex formation is proposed.

TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair.

TFIIH is a multisubunit protein complex involved in RNA polymerase II transcription and nucleotide excision repair, which removes a wide variety of DNA lesions including UV-induced photoproducts. Mutations in the DNA-dependent ATPase/helicase subunits of TFIIH, XPB and XPD, are associated with three inherited syndromes as follows: xeroderma pigmentosum with or without Cockayne syndrome and trichothiodystrophy. By using epitope-tagged XPD we purified mammalian TFIIH carrying a wild type or an active-site mutant XPD subunit. Contrary to XPB, XPD helicase activity was dispensable for in vitro transcription, catalytic formation of trinucleotide transcripts, and promoter opening. Moreover, in contrast to XPB, microinjection of mutant XPD cDNA did not interfere with in vivo transcription. These data show directly that XPD activity is not required for transcription. However, during DNA repair, neither 5' nor 3' incisions in defined positions around a DNA adduct were detected in the presence of TFIIH containing inactive XPD, although substantial damage-dependent DNA synthesis was induced by the presence of mutant XPD both in cells and cell extracts. The aberrant damage-dependent DNA synthesis caused by the mutant XPD does not lead to effective repair, consistent with the discrepancy between repair synthesis and survival in cells from a number of XP-D patients.

Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK.

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC-hHR23B, XPG, and ERCC1-XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase delta or epsilon, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

Distinct roles for the helicases of TFIIH in transcript initiation and promoter escape.

To provide an explanation of some clinical features observed within rare xeroderma pigmentosum (XP) patients and to further define the role of XPB, XPD, and cdk7, the three enzymatic subunits of TFIIH, in the transcription reaction, we have examined two defined enzymatic steps: phosphodiester bond formation and promoter escape. We provide evidence that the XPB helicase plays a dominant role in initiation, whereas the XPD helicase plays a minor contributing role in this step. The cyclin-activating kinase subcomplex of TFIIH improves the efficiency of initiation, but this involves only the structural contributions of cyclin-activating kinase rather than enzymatic activity. We demonstrate that XPB patient-derived mutants in TFIIH suffer from defects in initiation. Moreover, mutant analysis shows that in addition to its crucial role in initiation, the XPB helicase plays a critical enzymatic role in the promoter escape, whereas XPD plays an important structural role in the promoter escape process. Finally, using patient-derived mutations in TFIIH, we demonstrate deficiencies in promoter escape for both mutants of the class that suffer from combined xeroderma pigmentosum/Cockayne's syndrome.

TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism.

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH.

As part of TFIIH, XPB and XPD helicases have been shown to play a role in nucleotide excision repair (NER). Mutations in these subunits are associated with three genetic disorders: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and trichothiodystrophy (TTD). The strong heterogeneous clinical features observed in these patients cannot be explained by defects in NER alone. We decided to look at the transcriptional activity of TFIIH from cell lines of XP individuals. We set up an immunopurification procedure to isolate purified TFIIH from patient cell extracts. We demonstrated that mutations in two XP-B/CS patients decrease the transcriptional activity of the corresponding TFIIH by preventing promoter opening. The defect of XPB in transcription can be circumvented by artificial opening of the promoter. Western blot analysis and enzymatic assays indicate that XPD mutations affect the stoichiometric composition of TFIIH due to a weakness in the interaction between XPD-CAK complex and the core TFIIH, resulting in a partial reduction of transcription activity. This work, in addition to clarifying the role of the various TFIIH subunits, supports the current hypothesis that XP-B/D patients are more likely to suffer from transcription repair syndromes rather than DNA repair disorders alone.

Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7.

To understand the initiation of the transcription of protein-coding genes, we have dissected the role of the basal transcription/DNA repair factor TFIIH. Having succeeded in reconstituting a functionally active TFIIH from baculovirus recombinant polypeptides, we were able to analyze the role of XPB, XPD, and cdk7 subunits in the transcription reaction. Designing mutated recombinant subunits, we show that the XPB helicase is absolutely required for transcription to open the promoter around the start site whereas the XPD helicase, which is dispensable, stimulates transcription and allows the CAK complex to be anchored to TFIIH. In addition, we also show that cdk7 may phosphorylate the carboxy-terminal domain (CTD) of RNA pol II in the absence of promoter opening.

Mutations in the XPD helicase gene result in XP and TTD phenotypes, preventing interaction between XPD and the p44 subunit of TFIIH.

In most cases, xeroderma pigmentosum group D (XP-D) and trichothiodystrophy (TTD) patients carry mutations in the carboxy-terminal domain of the evolutionarily conserved helicase XPD, which is one of the subunits of the transcription/repair factor TFIIH (refs 1,2). In this study, we demonstrate that XPD interacts specifically with p44, another subunit of TFIIH, and that this interaction results in the stimulation of 5'-->3' helicase activity. Mutations in the XPD C-terminal domain, as found in most patients, prevent the interaction with p44, thus explaining the decrease in XPD helicase activity and the nucleotide excision repair (NER) defect.

TATA binding protein discriminates between different lesions on DNA, resulting in a transcription decrease.

DNA damage recognition by basal transcription factors follows different mechanisms. Using transcription-competition, nitrocellulose filter binding, and DNase I footprinting assays, we show that, although the general transcription factor TFIIH is able to target any kind of lesion which can be repaired by the nucleotide excision repair pathway, TATA binding protein (TBP)-TFIID is more selective in damage recognition. Only genotoxic agents which are able to induce kinked DNA structures similar to the one for the TATA box in its TBP complex are recognized. Indeed, DNase I footprinting patterns reveal that TBP protects equally 4 nucleotides upstream and 6 nucleotides downstream from the A-T (at position -29 of the noncoding strand) of the adenovirus major late promoter and from the G-G of a cisplatin-induced 1,2-d(GpG) cross-link. Together, our results may partially explain differences in transcription inhibition rates following DNA damage.

Affinity purification of human DNA repair/transcription factor TFIIH using epitope-tagged xeroderma pigmentosum B protein.

TFIIH is a high molecular weight complex with a remarkable dual function in nucleotide excision repair and initiation of RNA polymerase II transcription. Mutations in the largest subunits, the XPB and XPD helicases, are associated with three inherited disorders: xeroderma pigmentosum, Cockayne's syndrome, and trichothiodystrophy. To facilitate the purification and biochemical characterization of this intricate complex, we generated a cell line stably expressing tagged XPB, allowing the immunopurification of the XPB protein and associated factors. Addition of two tags, a N-terminal hexameric histidine stretch and a C-terminal hemagglutinin epitope, to this highly conserved protein did not interfere with its functioning in repair and transcription. The hemagglutinin epitope allowed efficient TFIIH immunopurification to homogeneity from a fractionated whole cell extract in essentially one step. We conclude that the predominant active form of TFIIH is composed of nine subunits and that there is one molecule of XPB per TFIIH complex. The affinity-purified complex exhibits all expected TFIIH activities: DNA-dependent ATPase, helicase, C-terminal domain kinase, and participation in in vitro and in vivo nucleotide excision repair and in vitro transcription. The affinity purification procedure described here is fast and simple, does not require extensive chromatographic procedures, and yields highly purified, active TFIIH.

DNA damage recognition by XPA protein promotes efficient recruitment of transcription factor II H.

The human basal transcription factor IIH (TFIIH) is an essential component of the nucleotide excision repair machinery. TFIIH is required for reaction steps concomitant with or prior to the formation of dual incisions in the damaged DNA strand. To understand the mechanism underlying the recruitment of TFIIH to DNA damage sites we have analyzed i) the direct affinity of TFIIH for damaged or undamaged DNA and ii) the interaction of TFIIH with XPA.DNA complexes, formed using unirradiated or UV-irradiated DNA. Filter binding assays showed that TFIIH has some affinity for the DNA, but in contrast to XPA, does not show any preference for UV-irradiated DNA. Pull-down experiments demonstrated that TFIIH binds to XPA.DNA complexes in an UV damage-dependent manner by a direct protein-protein interaction with XPA. We propose that an enhancement of the affinity of XPA protein for TFIIH could arise from conformational changes of XPA when it binds to UV lesions on the DNA.

Cisplatin- and UV-damaged DNA lure the basal transcription factor TFIID/TBP.

A connection between transcription and DNA repair was demonstrated previously through the characterization of TFIIH. Using filter binding as well as in vitro transcription challenge competition assays, we now show that the promoter recognition factor TATA box-binding protein (TBP)/TFIID binds selectively to and is sequestered by cisplatin- or UV-damaged DNA, either alone or in the context of a larger protein complex including TFIIH. Computer-assisted 3D structural analysis reveals a remarkable similarity between the structure of the TATA box as found in its TBP complex and that of either platinated or UV-damaged oligonucleotides. Thus, cisplatin-treated or UV-irradiated DNA could be used as a competing binding site which may lure TBP/TFIID away from its normal promoter sequence, partially explaining the phenomenon of DNA damage-induced inhibition of RNA synthesis. Consistent with an involvement of damaged DNA-specific binding of TBP in inhibiting transcription, we find that microinjection of additional TBP in living human fibroblasts alleviates the reduction in RNA synthesis after UV irradiation. Future anticancer drugs could be designed with the consideration of lesion recognition by TBP and their ability to reduce transcription.

Cellular strategies for accommodating replication-hindering adducts in DNA: control by the SOS response in Escherichia coli.

The replication of double-stranded plasmids containing a single adduct was analyzed in vivo by means of a sequence heterology that marks the two DNA strands. The single adduct was located within the sequence heterology, making it possible to distinguish trans-lesion synthesis (TLS) events from damage avoidance events in which replication did not proceed through the lesion. When the SOS system of the host bacteria is not induced, the C8-guanine adduct formed by the carcinogen N-2-acetylaminofluorene (AAF) yields less than 1% of TLS events, showing that replication does not readily proceed through the lesion. In contrast, the deacetylated adduct N-(deoxyguanosin-8-yl)-2-aminofluorene yields approximately 70% of TLS events under both SOS-induced and uninduced conditions. These results for TLS in vivo are in good agreement with the observation that AAF blocks DNA replication in vitro, whereas aminofluorene does so only weakly. Induction of the SOS response causes an increase in TLS events through the AAF adduct (approximately 13%). The increase in TLS is accompanied by a proportional increase in the frequency of AAF-induced frameshift mutations. However, the polymerase frameshift error rate per TLS event was essentially constant throughout the SOS response. In an SOS-induced delta umuD/C strain, both US events and mutagenesis are totally abolished even though there is no decrease in plasmid survival. Error-free replication evidently proceeds efficiently by means of the damage avoidance pathway. We conclude that SOS mutagenesis results from increased TLS rather than from an increased frameshift error rate of the polymerase.

Oxidation of reduced Cu,Zn superoxide dismutase by molecular oxygen. A kinetic study.

The rate of oxidation of reduced bovine Cu,Zn superoxide dismutase [ECu(I)] by molecular O2 was studied by magnetic-resonance techniques and was found to be low under physiological conditions. The analysis of the kinetic data and of the experiments carried out in the presence of tetranitromethane confirms that O2.- is a product of the oxidation process. At [ECu(I)]/([ECu(I)] + [ECu(II)]) greater than 0.5 the O2.- produced reacts mainly with ECu(I), increasing the oxidation rate of the enzyme, whereas at [ECu(I)]/([ECu(I)] + [ECu(II)]) less than 0.5 it reacts mainly with ECu(II), decreasing the oxidation rate, the kinetics, at constant O2 concentration, being an apparent second-order process. The oxidation rate increased linearly with both O2 and OH- concentration, indicating that only a deprotonated form of the ECu(I) reacts with O2.-.