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Schematic diagram of the X.borealis 5S rRNA gene fragment used in this study. The locations of Hin dIII (H) and Eco RI (E) restriction sites are shown. The arrow indicates the 5S rDNA, the direction of transcription and the transcription start site ( ϩ 1). The large open box represents the ICR of the 5S rDNA, and the smaller hatched boxes represent the positions of the A-box, IE and C-box, from left to right, respectively. The different fragment lengths are in base pairs (bp).
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UV-induced photoproducts (cyclobutane pyrimidine dimers, CPDs) in DNA are removed by nucleotide excision repair (NER), and the presence of transcription factors on DNA can restrict the accessibility of NER enzymes. We have investigatigated the modulation of NER in a gene promoter using the Xenopus transcription factor IIIA (TFIIIA)-5S rDNA complex...
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Context 1
... evaluate the modulation of NER by a tightly bound transcription factor, we followed repair of CPDs in the Xenopus borealis 5S rRNA gene complexed with transcrip- tion factor IIIA (TFIIIA). The 5S rRNA gene is 120 bp long and the minimal promoter element required for accurate initiation by RNA pol III is a 50 bp internal control region (ICR) (Wolffe, 1994; see Figure 1). The ICR is the binding site for TFIIIA, a 38 kDa protein composed of nine tandemly repeated zinc fingers and an additional C-terminal domain. ...
Context 2
... ICR is the binding site for TFIIIA, a 38 kDa protein composed of nine tandemly repeated zinc fingers and an additional C-terminal domain. Three elements were identified in the ICR: the A-box at the 5 end; the C-box at the 3 end; and the intermediate element (IE) between these boxes (Figure 1, shaded boxes). The three N-terminal zinc fingers of TFIIIA bind to the C-box (80 to 91), the three central zinc fingers bind to the IE (67 to 72), and the three C-terminal zinc fingers bind to the A-box (50 to 64). ...
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... cloned 214 bp fragment was used in these studies and contained all of the X.borealis 5S rRNA gene (120 bp), 60 bp of flanking DNA regions and 34 bp of plasmid DNA ( Liu et al., 1997; Figure 1). Since the conditions used for the formation of a stable TFIIIA-5S rDNA complex (Del Rio and Setzer, 1991;Liu et al., 1997) differ from those used for NER by the Xenopus oocyte nuclear extracts in vitro (Saxena et al., 1990;Oda et al., 1996), it was necessary to examine the stability of the complex during the repair reaction. ...
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... these data it is clear that CPDs in the TS disappear with increasing repair time ( Figure 6A, compare lanes 4 and 5 with lanes 6 to 13). Moreover, CPDs present outside the ICR are repaired at similar rates in both the naked rDNA and the TFIIIA-5S rDNA complex ( Figure 6A, compare lanes 6, 8, 10 and 12 with lanes 7, 9, 11 and 13, above and below the shaded bar). On the contrary, there is a striking difference between these samples in repair of CPDs inside the ICR (Figure 6A, Fig. 6. ...
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... DNA samples were treated with T4 endo V and separeted on denaturing gels as described in Figure 5. Lanes 1 to 13 are as in Figure 2. Shaded bar at right in each panel denotes the TFIIIA-binding site (ICR). Sites from -26 to 99 for the TS (A) and 96 to 3 for the NTS (B) denote the position (in bases) from the transcription start site (1; see Figure 1). sites within the shaded bar region). ...
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... and UV irradiation of the 5S rDNA fragment Plasmid pKS-5S ( Mann et al., 1997) was linearized with either EcoRI or HindIII (Life Technologies) to label the TS and the NTS, respectively. After dephosphorylation by alkaline phosphatase (Boehringer Mannheim), the linearized plasmid DNA was end-labeled with T4 polynucleotide kinase (Life Technologies) and digested with a second restriction enzyme (HindIII for TS-labeling and EcoRI for NTS-labeling), producing a single end-labeled 214 bp DNA fragment containing the X.borealis 5S rDNA sequence ( Liu et al., 1997; see Figure 1). After separation on a 2% preparative agarose gel, the end-labeled 214 bp DNA fragment was recovered and resuspended in TE buffer [10 mM Tris pH 7.5 and 1 mM EDTA], at a concentration of ~2 µg/ml. ...
Similar publications
Xenopus transcription factor IIIA (TFIIIA) binds to over 50 base pairs in the internal control region of the 5 S rRNA gene, yet the binding energy for this interaction (DeltaG0 = -12.8 kcal/mol) is no greater than that exhibited by many proteins that occupy much smaller DNA targets. Despite considerable study, the distribution of the DNA binding en...
We have used a circular permutation gel shift assay to show that the 5S gene transcription factor, TFIIIA, induces a bend
at the internal promoter of the Xenopus oocyte-type 5S gene. The degree of bending is comparable to what we have previously observed for TFIIIA induced bending of
the Xenopus somatic-type gene [Schroth, G.P. etal. (1989) Nature...
Functional deletion mutants of the trans-acting factor TFIIIA, truncated at both ends of the molecule, have been expressed
by in vitro transcription of a cDNA clone and subsequent cell-free translation of the synthetic mRNAs. A region of TFIIIA
19 amino acids or less, near the carboxyl terminus, is critical for maximal transcription and lies outsid...
The
Xenopus
TFIIIA gene is transcribed very efficiently in oocytes. In addition to a TATA element at −30, we show that from −425 to +7 the TFIIIA gene contains only two positive
cis
elements centered at −267 (element 1) and −230 (element 2). This arrangement of the
cis
elements in the TFIIIA gene is striking because these two elements are positione...
Transcription factor IIIA (TFIIIA), a sequence-specific DNA-binding protein from Xenopus laevis, is a zinc finger protein required for transcription of 5S rRNA genes by RNA polymerase III. We describe the purification
and characterization of recombinant TFIIIA (recTFIIIA) expressed in E. coli. RecTFIIIA was purified to greater than 95% homogeneity...
Citations
... Specifically, recent genomic studies showed that nucleotide excision repair (NER) is less efficient at active TF binding sites, while mutation rates at these sites are significantly elevated, consistent with the hypothesis that TFs impair repair and increase mutagenesis at their binding sites (13,14). Although the exact mechanisms by which specific TFs influence mutagenesis are not fully understood, there is growing evidence that TFs might compete with DNA repair proteins for the damaged DNA substrate, and thus effectively act as a barrier for the repair machinery (14)(15)(16). This competition, though, depends strongly on whether TFs can still bind efficiently to their target genomic DNA sites in the presence of UV lesions. ...
Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hypermutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all the TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs, we identified a surprising but reproducible effect at certain nonconsensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and nonconsensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
... Specifically, recent genomic studies showed that nucleotide excision repair (NER) is less efficient at active TF binding sites, while mutation rates at these sites are significantly elevated, consistent with the hypothesis that TFs impair repair and increase mutagenesis at their binding sites (12,13). Although the exact mechanisms by which specific TFs influence mutagenesis are not fully understood, there is growing evidence that TFs might compete with DNA repair proteins for the damaged DNA substrate, and thus effectively act as a barrier for the repair machinery (13)(14)(15). This competition, though, depends strongly on whether TFs can still bind efficiently to their target genomic DNA sites in the presence of UV lesions. ...
Somatic mutations are highly enriched at transcription factor (TF) binding sites, with the strongest trend being observed for ultraviolet light (UV)-induced mutations in melanomas. One of the main mechanisms proposed for this hyper-mutation pattern is the inefficient repair of UV lesions within TF-binding sites, caused by competition between TFs bound to these lesions and the DNA repair proteins that must recognize the lesions to initiate repair. However, TF binding to UV-irradiated DNA is poorly characterized, and it is unclear whether TFs maintain specificity for their DNA sites after UV exposure. We developed UV-Bind, a high-throughput approach to investigate the impact of UV irradiation on protein-DNA binding specificity. We applied UV-Bind to ten TFs from eight structural families, and found that UV lesions significantly altered the DNA-binding preferences of all TFs tested. The main effect was a decrease in binding specificity, but the precise effects and their magnitude differ across factors. Importantly, we found that despite the overall reduction in DNA-binding specificity in the presence of UV lesions, TFs can still compete with repair proteins for lesion recognition, in a manner consistent with their specificity for UV-irradiated DNA. In addition, for a subset of TFs we identified a surprising but reproducible effect at certain non-consensus DNA sequences, where UV irradiation leads to a high increase in the level of TF binding. These changes in DNA-binding specificity after UV irradiation, at both consensus and non-consensus sites, have important implications for the regulatory and mutagenic roles of TFs in the cell.
... While TFs mainly function in transcriptional regulation, their binding to DNA can affect DNA damage formation and repair (Mao and Wyrick, 2019). To this end, several TF proteins have been shown to modulate formation of ultraviolet (UV) light-induced photolesions (Frigola et al., 2021;Hu et al., 2017;Mao et al., 2018) and inhibit nucleotide excision repair (NER) (Conconi et al., 1999;Sabarinathan et al., 2016). The altered UV damage formation and suppressed NER are believed to cause increased mutation rates at TF binding sites in skin cancers (Frigola et al., 2021;Mao et al., 2018;Sabarinathan et al., 2016). ...
DNA base damage arises frequently in living cells and needs to be removed by base excision repair (BER) to prevent mutagenesis and genome instability. Both the formation and repair of base damage occur in chromatin and are conceivably affected by DNA-binding proteins such as transcription factors (TFs). However, to what extent TF binding affects base damage distribution and BER in cells is unclear. Here, we used a genome-wide damage mapping method, N -methylpurine-sequencing (NMP-seq), and characterized alkylation damage distribution and BER at TF binding sites in yeast cells treated with the alkylating agent methyl methanesulfonate (MMS). Our data shows that alkylation damage formation was mainly suppressed at the binding sites of yeast TFs Abf1 and Reb1, but individual hotspots with elevated damage levels were also found. Additionally, Abf1 and Reb1 binding strongly inhibits BER in vivo and in vitro , causing slow repair both within the core motif and its adjacent DNA. Repair of UV damage by nucleotide excision repair (NER) was also inhibited by TF binding. Interestingly, TF binding inhibits a larger DNA region for NER relative to BER. The observed effects are caused by the TF-DNA interaction, because damage formation and BER can be restored by depletion of Abf1 or Reb1 protein from the nucleus. Thus, our data reveal that TF binding significantly modulates alkylation base damage formation and inhibits repair by the BER pathway. The interplay between base damage formation and BER may play an important role in affecting mutation frequency in gene regulatory regions.
... Thus, depending on the position of PDs on kinked DNA around the protein, their recognition can occur at different rate. This has been shown for gene promoters and nucleosomal DNA (41,58,59). ...
... Quantitative analyses showed that for the WT strain the percent of CPD removed after 1, 2 and 4 h repair were about 37, 49 and 69%. Similar NER efficiencies were found for sir2Δ (35,52 and 83%) and for sir3Δ (36,58 and 85%). Additional information was obtained after digestion with XhoI (Supplementary Figure S1C) that released a population of fragments (broad band) with an average length of ∼1.3 kb (Figure 5C), of which ∼27% was made of telomeric Nucleic Acids Research, 2017, Vol. ...
Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent.
... In general, repair of UV photoproducts in promoter DNA elements is inhibited by the presence of transcription factors (7,(40)(41)(42)(43), and the results show that repair is slow over the entire RNAP1 promoter and downstream of the transcription initiation site. The plausible explanation is that PIC obstructs DNA damage recognition and repair up to nucleotide +34, since NER covers a space of about 30 nucleotides around the damaged site (44). ...
If not repaired, ultraviolet light induced DNA damage can lead to genome instability. Nucleotide excision repair (NER) of UV photoproducts is generally fast in the coding region of genes, where RNA polymerase-II (RNAP2) arrest at damage sites and trigger transcription-coupled NER (TC-NER). In Saccharomyces cerevisiae there is RNA polymerase-I (RNAP1) dependent TC-NER, but this process remains elusive. Therefore, we wished to characterize TC-NER efficiency in different regions of the rDNA locus: where RNAP1 are present at high density and start transcription elongation, where the elongation rate is slow, and in the transcription terminator where RNAP1 pause, accumulate and then are released. The Rpa12 subunit of RNAP1 and the Nsi1 protein participate in transcription termination, and NER efficiency was compared between wild type and cells lacking Rpa12 or Nsi1. The presence of RNAP1 was determined by chromatin endogenous cleavage and chromatin immuno-precipitation, repair was followed at nucleotide precision with an assay that is based on the blockage of Taq polymerase by UV photoproducts. We describe that TC-NER, which is modulated by the RNAP1 level and elongation rate, ends at the 35S rRNA gene transcription termination-site. This article is protected by copyright. All rights reserved.
... As expected, C > T (G> A) mutations predominated in melanomas over other nucleotide changes (Fig. 1d), both within TFBS and at their flanks. This could be explained by either a faulty DNA repair 7,8 or a higher probability of UV induced lesions 22,23 at protein-bound DNA. ...
Somatic mutations are the driving force of cancer genome evolution. The rate of somatic mutations appears to be greatly variable across the genome due to variations in chromatin organization, DNA accessibility and replication timing. However, other variables that may influence the mutation rate locally are unknown, such as a role for DNA-binding proteins, for example. Here we demonstrate that the rate of somatic mutations in melanomas is highly increased at active transcription factor binding sites and nucleosome embedded DNA, compared to their flanking regions. Using recently available excision-repair sequencing (XR-seq) data, we show that the higher mutation rate at these sites is caused by a decrease of the levels of nucleotide excision repair (NER) activity. Our work demonstrates that DNA-bound proteins interfere with the NER machinery, which results in an increased rate of DNA mutations at the protein binding sites. This finding has important implications for our understanding of mutational and DNA repair processes and in the identification of cancer driver mutations.
... Similar repair efficiency was measured for the coding and flanking DNA sequences, while a number of dimers in the 5 -flanking sequences of the tRNA Val gene were repaired faster than in the coding region [114]. The presence of transcription factors III-C and III-B might have impeded DNA damage recognition and repair, as later shown for the TFIIIA-5S rRNA gene complex in vitro [115]. In addition, TC-NER that is characterized by faster removal of DNA lesions from the TS than from the NTS was not observed in the two tRNA genes. ...
... Singh and Vadåsz reported that chain breaks in ribosomal RNA have been observed in inactive ribosomes of gamma irradiated-Escherichia coli [60]. Conconi et al. reported that UV irradiation caused ribosomal DNA damage and described repair-independent chromatin assembly onto active ribosomal genes after UV irradiation [61]. Under our experimental conditions, induction of these genes may reflect DNA damage of the yeast ribosome by fast neutron irradiation. ...
To understand the yeast response to high-linear energy transfer (LET) ionizing radiation (IR), we investigated global gene expression in yeast irradiated by three types of high-LET IR (fast neutrons, heavy ions, and thermal neutrons) and gamma rays using DNA microarray analysis. Stationary cells were irradiated by each IR and recultured in yeast-peptone-dextrose medium to allow repair for 40 min. RNA was then isolated from three independent samples of irradiated yeast. Genes involved in the Mec1p kinase pathway, which functions in DNA damage response, were induced by all forms of high-LET IR and by gamma rays. Some genes related to oxidative stress and the cell wall were induced by all forms of high-LET IRs. Gene expression patterns as a function of each type of high-LET IR were examined statistically by one-way analysis of variance. This analysis demonstrated the existence of irradiation-specific responses. For example, genes involved in ribosomal DNA synthesis were specifically induced by fast neutron irradiation, while the ubiquitin-proteasome system and heat shock response were specifically induced by thermal neutron irradiation. The study characterizes high-LET IR-induced gene expression and provides a molecular understanding of subsequent adaptation in yeast.
... Large bulky adducts between adjoining nucleotides have been shown to physically block access of proteins necessary for cellular maintenance such as replication and transcription (2). Indeed, we have previously shown that binding of transcription factor IIIA to the 5 S rRNA gene decreases the rate of repair of UV radiation-induced cyclobutane pyrimidine dimers located in the internal control region (3). Furthermore, introduction of single cyclobutane pyrimidine dimer lesions at six different sites in the internal control region of 5 S rDNA allowed us to map the binding strength of transcription factor IIIA to its cognate sequence (4,5). ...
DNA repair takes place in the context of chromatin. Previous studies showed that histones impair base excision repair (BER)
of modified bases at both the excision and synthesis steps. We examined BER of uracil in a glucocorticoid response element
(GRE) complexed with the glucocorticoid receptor DNA binding domain (GR-DBD). Five substrates were designed, each containing
a unique C→U substitution within the mouse mammary tumor virus promoter, one located within each GRE half-site and the others
located outside the GRE. To examine distinct steps of BER, DNA cleavage by uracil-DNA glycosylase and Ape1 endonuclease was
used to assess initiation, dCTP incorporation by DNA polymerase (pol) β was used to measure repair synthesis, and DNA ligase
I was used to seal the nick. For uracil sites within the GRE, there was a reduced rate of uracil-DNA glycosylase/Ape1 activity
following GR-DBD binding. Cleavage in the right half-site, with higher GR-DBD binding affinity, was reduced ∼5-fold, whereas
cleavage in the left half-site was reduced ∼3.8-fold. Conversely, uracil-directed cleavage outside the GRE was unaffected
by GR-DBD binding. Surprisingly, there was no reduction in the rate of pol β synthesis or DNA ligase activity on any of the
fragments bound to GR-DBD. Indeed, we observed a small increase (∼1.5–2.2-fold) in the rate of pol β synthesis at uracil residues
in both the GRE and one site six nucleotides downstream. These results highlight the potential for both positive and negative impacts of DNA-transcription factor binding on the rate of BER.
... In WT cells, NER is slow over the entire RNAPI promoter and downstream of the transcription initiation site up to nucleotide ϩ34 (Fig. 4C). Similarly, NER is slow in the RNAPII and -III promoters (9,50). Thus, it is likely that RNA polymerase transcription initiation complexes obstruct the multienzymatic mechanism of NER, which covers about 30 nucleotides around the damaged site (62). ...
Nucleotide excision repair (NER) removes a plethora of DNA lesions. It is performed by a large multisubunit protein complex
that finds and repairs damaged DNA in different chromatin contexts and nuclear domains. The nucleolus is the most transcriptionally
active domain, and in yeast, transcription-coupled NER occurs in RNA polymerase I-transcribed genes (rDNA). Here we have analyzed
the roles of two members of the xeroderma pigmentosum group C family of proteins, Rad4p and Rad34p, during NER in the active
and inactive rDNA. We report that Rad4p is essential for repair in the intergenic spacer, the inactive rDNA coding region,
and for strand-specific repair at the transcription initiation site, whereas Rad34p is not. Rad34p is necessary for transcription-coupled
NER that starts about 40 nucleotides downstream of the transcription initiation site of the active rDNA, whereas Rad4p is
not. Thus, although Rad4p and Rad34p share sequence homology, their roles in NER in the rDNA locus are almost entirely distinct
and complementary. These results provide evidences that transcription-coupled NER and global genome NER participate in the
removal of UV-induced DNA lesions from the transcribed strand of active rDNA. Furthermore, nonnucleosome rDNA is repaired
faster than nucleosome rDNA, indicating that an open chromatin structure facilitates NER in vivo.
