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2.
Genetics ; 206(1): 179-187, 2017 05.
Article in English | MEDLINE | ID: mdl-28341651

ABSTRACT

We made a coupled genetic reporter that detects rare transcription misincorporation errors to measure RNA polymerase transcription fidelity in Escherichia coli Using this reporter, we demonstrated in vivo that the transcript cleavage factor GreA, but not GreB, is essential for proofreading of a transcription error where a riboA has been misincorporated instead of a riboG. A greA mutant strain had more than a 100-fold increase in transcription errors relative to wild-type or a greB mutant. However, overexpression of GreB in ΔgreA cells reduced the misincorporation errors to wild-type levels, demonstrating that GreB at high concentration could substitute for GreA in RNA proofreading activity in vivo.


Subject(s)
Escherichia coli Proteins/genetics , Genes, Reporter/genetics , Transcription Factors/genetics , Transcription, Genetic , Transcriptional Elongation Factors/genetics , DNA-Directed RNA Polymerases/genetics , Escherichia coli/genetics , Gene Expression Regulation, Bacterial , Peptide Elongation Factors , Promoter Regions, Genetic , RNA/biosynthesis , RNA/genetics
3.
Proc Natl Acad Sci U S A ; 113(44): 12426-12431, 2016 11 01.
Article in English | MEDLINE | ID: mdl-27791148

ABSTRACT

RNA polymerase II (pol II) encounters numerous barriers during transcription elongation, including DNA strand breaks, DNA lesions, and nucleosomes. Pyrrole-imidazole (Py-Im) polyamides bind to the minor groove of DNA with programmable sequence specificity and high affinity. Previous studies suggest that Py-Im polyamides can prevent transcription factor binding, as well as interfere with pol II transcription elongation. However, the mechanism of pol II inhibition by Py-Im polyamides is unclear. Here we investigate the mechanism of how these minor-groove binders affect pol II transcription elongation. In the presence of site-specifically bound Py-Im polyamides, we find that the pol II elongation complex becomes arrested immediately upstream of the targeted DNA sequence, and is not rescued by transcription factor IIS, which is in contrast to pol II blockage by a nucleosome barrier. Further analysis reveals that two conserved pol II residues in the Switch 1 region contribute to pol II stalling. Our study suggests this motif in pol II can sense the structural changes of the DNA minor groove and can be considered a "minor groove sensor." Prolonged interference of transcription elongation by sequence-specific minor groove binders may present opportunities to target transcription addiction for cancer therapy.


Subject(s)
DNA/metabolism , Nylons/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic/genetics , Amino Acid Sequence , Binding Sites/genetics , DNA/chemistry , DNA/genetics , Imidazoles/chemistry , Imidazoles/metabolism , Imidazoles/pharmacology , Models, Molecular , Nucleic Acid Conformation , Nylons/chemistry , Nylons/pharmacology , Protein Binding/drug effects , Protein Domains , Pyrroles/chemistry , Pyrroles/metabolism , Pyrroles/pharmacology , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/genetics , Sequence Homology, Amino Acid , Transcription, Genetic/drug effects
5.
Nat Commun ; 6: 8065, 2015 Aug 25.
Article in English | MEDLINE | ID: mdl-26304740

ABSTRACT

Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.


Subject(s)
Cellular Senescence/genetics , Molecular Chaperones/metabolism , Protein Aggregation, Pathological/metabolism , Stress, Physiological , Transcription, Genetic , Cell Line , Cell Survival/genetics , Heat-Shock Proteins/metabolism , Mutation , RNA Polymerase II/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism
6.
PLoS Genet ; 11(1): e1004910, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25569256

ABSTRACT

Mutations accumulate during all stages of growth, but only germ line mutations contribute to evolution. While meiosis contributes to evolution by reassortment of parental alleles, we show here that the process itself is inherently mutagenic. We have previously shown that the DNA synthesis associated with repair of a double-strand break is about 1000-fold less accurate than S-phase synthesis. Since the process of meiosis involves many programmed DSBs, we reasoned that this repair might also be mutagenic. Indeed, in the early 1960's Magni and Von Borstel observed elevated reversion of recessive alleles during meiosis, and found that the revertants were more likely to be associated with a crossover than non-revertants, a process that they called "the meiotic effect." Here we use a forward mutation reporter (CAN1 HIS3) placed at either a meiotic recombination coldspot or hotspot near the MAT locus on Chromosome III. We find that the increased mutation rate at CAN1 (6 to 21 -fold) correlates with the underlying recombination rate at the locus. Importantly, we show that the elevated mutation rate is fully dependent upon Spo11, the protein that introduces the meiosis specific DSBs. To examine associated recombination we selected for random spores with or without a mutation in CAN1. We find that the mutations isolated this way show an increased association with recombination (crossovers, loss of crossover interference and/or increased gene conversion tracts). Polζ appears to contribute about half of the mutations induced during meiosis, but is not the only source of mutations for the meiotic effect. We see no difference in either the spectrum or distribution of mutations between mitosis and meiosis. The correlation of hotspots with elevated mutagenesis provides a mechanism for organisms to control evolution rates in a gene specific manner.


Subject(s)
Meiosis/genetics , Mitosis/genetics , Mutation Rate , Recombination, Genetic , Amino Acid Transport Systems, Basic/genetics , Chromosomes/metabolism , DNA Repair/genetics , Endodeoxyribonucleases/genetics , Gene Conversion/genetics , Homeodomain Proteins/genetics , Mutation , Repressor Proteins/genetics , Saccharomyces cerevisiae , Saccharomyces cerevisiae Proteins/genetics
7.
PLoS Genet ; 10(9): e1004532, 2014 Sep.
Article in English | MEDLINE | ID: mdl-25232834

ABSTRACT

We developed a highly sensitive assay to detect transcription errors in vivo. The assay is based on suppression of a missense mutation in the active site tyrosine in the Cre recombinase. Because Cre acts as tetramer, background from translation errors are negligible. Functional Cre resulting from rare transcription errors that restore the tyrosine codon can be detected by Cre-dependent rearrangement of reporter genes. Hence, transient transcription errors are captured as stable genetic changes. We used this Cre-based reporter to screen for mutations of Saccharomyces cerevisiae RPB1 (RPO21) that increase the level of misincorporation during transcription. The mutations are in three domains of Rpb1, the trigger loop, the bridge helix, and in sites involved in binding to TFIIS. Biochemical characterization demonstrates that these variants have elevated misincorporation, and/or ability to extend mispaired bases, or defects in TFIIS mediated editing.


Subject(s)
RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Transcription, Genetic/genetics , Amino Acid Sequence , Catalytic Domain/genetics , Codon/genetics , Gene Expression Regulation, Fungal/genetics , Genes, Reporter/genetics , Molecular Sequence Data , Mutation/genetics
8.
J Mol Biol ; 425(4): 697-712, 2013 Feb 22.
Article in English | MEDLINE | ID: mdl-23238253

ABSTRACT

Pausing of RNA polymerase II (RNAP II) by backtracking on DNA is a major regulatory mechanism in control of eukaryotic transcription. Backtracking occurs by extrusion of the 3' end of the RNA from the active center after bond formation and before translocation of RNAP II on DNA. In several documented cases, backtracking requires a special signal such as A/T-rich sequences forming an unstable RNA-DNA hybrid in the elongation complex. However, other sequence-dependent backtracking signals and conformations of RNAP II leading to backtracking remain unknown. Here, we demonstrate with S. cerevisiae RNAP II that a cleavage-deficient elongation factor TFIIS (TFIIS(AA)) enhances backtracked pauses during regular transcription. This is due to increased efficiency of formation of an intermediate that leads to backtracking. This intermediate may involve misalignment at the 3' end of the nascent RNA in the active center of the yeast RNAP II, and TFIIS(AA) promotes formation of this intermediate at the DNA sequences, presenting a high-energy barrier to translocation. We proposed a three-step mechanism for RNAP II pausing in which a prolonged dwell time in the pre-translocated state increases the likelihood of the 3' RNA end misalignment facilitating a backtrack pausing. These results demonstrate an important role of the intrinsic blocks to forward translocation in pausing by RNAP II.


Subject(s)
RNA Polymerase II/metabolism , RNA, Fungal/genetics , Saccharomyces cerevisiae Proteins/metabolism , Transcription, Genetic , Base Sequence , DNA, Fungal/chemistry , DNA, Fungal/genetics , Kinetics , Models, Genetic , Mutation , Protein Transport , RNA Polymerase II/genetics , RNA, Fungal/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Transcriptional Elongation Factors/genetics , Transcriptional Elongation Factors/metabolism
9.
Mol Cell ; 46(1): 18-29, 2012 Apr 13.
Article in English | MEDLINE | ID: mdl-22405652

ABSTRACT

UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA strand stall transcription elongation by RNA polymerase II (Pol II). If the nucleotide excision repair machinery does not promptly remove the CPDs, stalled Pol II creates a roadblock for DNA replication and subsequent rounds of transcription. Here we present evidence that Pol II has an intrinsic capacity for translesion synthesis (TLS) that enables bypass of the CPD with or without repair. Translesion synthesis depends on the trigger loop and bridge helix, the two flexible regions of the Pol II subunit Rpb1 that participate in substrate binding, catalysis, and translocation. Substitutions in Rpb1 that promote lesion bypass in vitro increase UV resistance in vivo, and substitutions that inhibit lesion bypass decrease cell survival after UV irradiation. Thus, translesion transcription becomes essential for cell survival upon accumulation of the unrepaired CPD lesions in genomic DNA.


Subject(s)
DNA Damage/radiation effects , Pyrimidine Dimers/metabolism , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription, Genetic/radiation effects , Ultraviolet Rays/adverse effects , DNA Replication/genetics , DNA Replication/radiation effects , DNA, Fungal/biosynthesis , DNA, Fungal/genetics , Genome, Fungal/physiology , Pyrimidine Dimers/genetics , RNA Polymerase II/genetics , Radiation Tolerance/genetics , Radiation Tolerance/radiation effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription, Genetic/genetics
10.
Biochim Biophys Acta ; 1819(7): 694-9, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22366339

ABSTRACT

Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.


Subject(s)
Escherichia coli Proteins/genetics , Mutation , RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/genetics , Transcription, Genetic , Amino Acid Motifs , Base Sequence , Escherichia coli Proteins/metabolism , Escherichia coli Proteins/physiology , Humans , RNA Polymerase II/metabolism , RNA Polymerase II/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/physiology
11.
J Biol Chem ; 284(29): 19601-12, 2009 Jul 17.
Article in English | MEDLINE | ID: mdl-19439405

ABSTRACT

Rpb9 is a small non-essential subunit of yeast RNA polymerase II located on the surface on the enzyme. Deletion of the RPB9 gene shows synthetic lethality with the low fidelity rpb1-E1103G mutation localized in the trigger loop, a mobile element of the catalytic Rpb1 subunit, which has been shown to control transcription fidelity. Similar to the rpb1-E1103G mutation, the RPB9 deletion substantially enhances NTP misincorporation and increases the rate of mismatch extension with the next cognate NTP in vitro. Using pre-steady state kinetic analysis, we show that RPB9 deletion promotes sequestration of NTPs in the polymerase active center just prior to the phosphodiester bond formation. We propose a model in which the Rpb9 subunit controls transcription fidelity by delaying the closure of the trigger loop on the incoming NTP via interaction between the C-terminal domain of Rpb9 and the trigger loop. Our findings reveal a mechanism for regulation of transcription fidelity by protein factors located at a large distance from the active center of RNA polymerase II.


Subject(s)
Fungal Proteins/metabolism , Nucleotides/metabolism , RNA Polymerase II/metabolism , Transcription, Genetic , Adenosine Triphosphate/metabolism , Base Sequence , Cytidine Triphosphate/metabolism , Fungal Proteins/genetics , Kinetics , Models, Molecular , Mutation , Protein Conformation , Protein Subunits/chemistry , Protein Subunits/genetics , Protein Subunits/metabolism , RNA Polymerase II/chemistry , RNA Polymerase II/genetics , Time Factors , Uridine Triphosphate/metabolism
12.
Mol Cell ; 30(5): 557-66, 2008 Jun 06.
Article in English | MEDLINE | ID: mdl-18538654

ABSTRACT

To study fidelity of RNA polymerase II (Pol II), we analyzed properties of the 6-azauracil-sensitive and TFIIS-dependent E1103G mutant of rbp1 (rpo21), the gene encoding the catalytic subunit of Pol II in Saccharomyces cerevisiae. Using an in vivo retrotransposition-based transcription fidelity assay, we observed that rpb1-E1103G causes a 3-fold increase in transcription errors. This mutant showed a 10-fold decrease in fidelity of transcription elongation in vitro. The mutation does not appear to significantly affect translocation state equilibrium of Pol II in a stalled elongation complex. Primarily, it promotes NTP sequestration in the polymerase active center. Furthermore, pre-steady-state analyses revealed that the E1103G mutation shifted the equilibrium between the closed and the open active center conformations toward the closed form. Thus, open conformation of the active center emerges as an intermediate essential for preincorporation fidelity control. Similar mechanisms may control fidelity of DNA-dependent DNA polymerases and RNA-dependent RNA polymerases.


Subject(s)
Gene Expression Regulation, Fungal , Mutation/genetics , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/enzymology , Transcription, Genetic , Amino Acid Sequence , Amino Acid Substitution , Binding Sites , Catalytic Domain , Isomerism , Molecular Sequence Data , Nucleotides/metabolism , RNA Polymerase II/genetics , Retroelements/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Sequence Alignment , Substrate Specificity
13.
Microbiol Res ; 163(1): 113-9, 2008.
Article in English | MEDLINE | ID: mdl-17923397

ABSTRACT

Hexavalent chromium is known to be a potent carcinogen that leads to many different DNA lesions, including DNA-protein crosslinks, and single- and double-strand breaks. In Saccharomyces cerevisiae, DNA double-strand breaks are mainly repaired by either homologous recombination (HR) or non-homologous end-joining (NHEJ) repair pathways. Here, we show that mutants deficient in NHEJ (yku70Delta, rad50Delta, dnl4Delta, mre11Delta, xrs2Delta) of S. cerevisiae are more sensitive to Cr(VI) toxic effects than wild-type cells. Also, a deletion mutant of SAE2 showed a similar sensitivity to Cr(VI), even though it has no apparent direct role in NHEJ. We also found that double mutants in HR and NHEJ (yku70Delta/rad52Delta, rad50Delta/rad52Delta, dnl4Delta/rad52Delta, mre11Delta/rad52Delta, xrs2Delta/rad52Delta) are synergistically more sensitive to Cr(VI) exposure than any of the single mutants, indicating that both repair pathways are involved in the repair of Cr(VI)-induced lesions. Finally, when the NHEJ mutants were exposed to Cr(VI) under anaerobic growth conditions, Cr(VI) toxicity was suppressed.


Subject(s)
Chromium/adverse effects , DNA Breaks, Double-Stranded , DNA Repair/genetics , DNA, Fungal/genetics , Recombination, Genetic , Saccharomyces cerevisiae/genetics , Anaerobiosis , Chromosome Breakage , Endonucleases , Mutation , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae Proteins/genetics
14.
Genetics ; 172(4): 2201-9, 2006 Apr.
Article in English | MEDLINE | ID: mdl-16510790

ABSTRACT

RNA polymerase II (RNAPII) in eukaryotic cells drives transcription of most messenger RNAs. RNAPII core enzyme is composed of 12 polypeptides where Rpb1 is the largest subunit. To further understand the mechanisms of RNAPII transcription, we isolated and characterized novel point mutants of RPB1 that are sensitive to the nucleotide-depleting drug 6-azauracil (6AU). In this work we reisolated the rpo21-24/rpb1-E1230K allele, which reduces the interaction of RNAPII-TFIIS, and identified five new point mutations in RPB1 that cause hypersensitivity to 6AU. The novel mutants affect highly conserved residues of Rpb1 and have differential genetic and biochemical effects. Three of the mutations affect the "lid" and "rudder," two small loops suggested by structural studies to play a central role in the separation of the RNA-DNA hybrids. Most interestingly, two mutations affecting the catalytic center (rpb1-N488D) and the homology box G (rpb1-E1103G) have strong opposite effects on the intrinsic in vitro polymerization rate of RNAPII. Moreover, the synthetic interactions of these mutants with soh1, spt4, and dst1 suggest differential in vivo effects.


Subject(s)
Mutation , RNA Polymerase II/genetics , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae/genetics , Uracil/analogs & derivatives , Amino Acid Sequence , Antimetabolites/pharmacology , Catalytic Domain , DNA/chemistry , Molecular Sequence Data , Peptides/chemistry , Point Mutation , RNA/chemistry , RNA Polymerase II/chemistry , RNA Polymerase II/metabolism , RNA, Messenger/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Sequence Homology, Amino Acid , Uracil/pharmacology
15.
CSH Protoc ; 2006(1)2006 Jun 01.
Article in English | MEDLINE | ID: mdl-22485546
17.
CSH Protoc ; 2006(1)2006 Jun 01.
Article in English | MEDLINE | ID: mdl-22485548
18.
CSH Protoc ; 2006(1)2006 Jun 01.
Article in English | MEDLINE | ID: mdl-22485549
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