RESUMO
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.
Assuntos
Dano ao DNA/efeitos da radiação , Dímeros de Pirimidina/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Replicação do DNA/genética , Replicação do DNA/efeitos da radiação , DNA Fúngico/biossíntese , DNA Fúngico/genética , Genoma Fúngico/fisiologia , Dímeros de Pirimidina/genética , RNA Polimerase II/genética , Tolerância a Radiação/genética , Tolerância a Radiação/efeitos da radiação , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica/genéticaRESUMO
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.
Assuntos
DNA/metabolismo , Nylons/metabolismo , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica/genética , Sequência de Aminoácidos , Sítios de Ligação/genética , DNA/química , DNA/genética , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Modelos Moleculares , Conformação de Ácido Nucleico , Nylons/química , Nylons/farmacologia , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Pirróis/química , Pirróis/metabolismo , Pirróis/farmacologia , RNA Polimerase II/química , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transcrição Gênica/efeitos dos fármacosRESUMO
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.
Assuntos
Meiose/genética , Mitose/genética , Taxa de Mutação , Recombinação Genética , Sistemas de Transporte de Aminoácidos Básicos/genética , Cromossomos/metabolismo , Reparo do DNA/genética , Endodesoxirribonucleases/genética , Conversão Gênica/genética , Proteínas de Homeodomínio/genética , Mutação , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Transcrição Gênica/genética , Sequência de Aminoácidos , Domínio Catalítico/genética , Códon/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Reporter/genética , Dados de Sequência Molecular , Mutação/genéticaRESUMO
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.
Assuntos
Regulação Fúngica da Expressão Gênica , Mutação/genética , RNA Polimerase II/química , RNA Polimerase II/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Transcrição Gênica , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Domínio Catalítico , Isomerismo , Dados de Sequência Molecular , Nucleotídeos/metabolismo , RNA Polimerase II/genética , Retroelementos/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
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.
Assuntos
Proteínas de Escherichia coli/genética , Mutação , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcrição Gênica , Motivos de Aminoácidos , Sequência de Bases , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Humanos , RNA Polimerase II/metabolismo , RNA Polimerase II/fisiologia , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
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.
Assuntos
Cromo/efeitos adversos , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA Fúngico/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Anaerobiose , Quebra Cromossômica , Endonucleases , Mutação , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
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.
Assuntos
Mutação , RNA Polimerase II/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Uracila/análogos & derivados , Sequência de Aminoácidos , Antimetabólitos/farmacologia , Domínio Catalítico , DNA/química , Dados de Sequência Molecular , Peptídeos/química , Mutação Puntual , RNA/química , RNA Polimerase II/química , RNA Polimerase II/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Homologia de Sequência de Aminoácidos , Uracila/farmacologiaRESUMO
The nature of any long palindrome that might exist in the human genome is obscured by the instability of such sequences once cloned in Escherichia coli. We describe and validate a practical alternative to the analysis of naturally-occurring palindromes based upon cloning and propagation in Saccharomyces cerevisiae. With this approach we have investigated an intronic sequence in the human Neurofibromatosis 1 (NF1) locus that is represented by multiple conflicting versions in GenBank. We find that the site is highly polymorphic, exhibiting different degrees of palindromy in different individuals. A side-by-side comparison of the same plasmids in E.coli versus. S.cerevisiae demonstrated that the more palindromic alleles were inevitably corrupted upon cloning in E.coli, but could be propagated intact in yeast. The high quality sequence obtained from the yeast-based approach provides insight into the various mechanisms that destabilize a palindrome in E.coli, yeast and humans, into the diversification of a highly polymorphic site within the NF1 locus during primate evolution, and into the association between palindromy and chromosomal translocation.
Assuntos
Genes da Neurofibromatose 1 , Genoma Humano , Genômica/métodos , Íntrons , Polimorfismo Genético , Sequências Repetitivas de Ácido Nucleico , Alelos , Artefatos , Sequência de Bases , Clonagem Molecular , Escherichia coli/genética , Evolução Molecular , Humanos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da Polimerase , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Deleção de SequênciaRESUMO
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.
Assuntos
Proteínas de Escherichia coli/genética , Genes Reporter/genética , Fatores de Transcrição/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Fatores de Alongamento de Peptídeos , Regiões Promotoras Genéticas , RNA/biossíntese , RNA/genéticaRESUMO
TFIIS promotes the intrinsic ability of RNA polymerase II to cleave the 3'-end of the newly synthesized RNA. This stimulatory activity of TFIIS, which is dependent upon Rpb9, facilitates the resumption of transcription elongation when the polymerase stalls or arrests. While TFIIS has a pronounced effect on transcription elongation in vitro, the deletion of DST1 has no major effect on cell viability. In this work we used a genetic approach to increase our knowledge of the role of TFIIS in vivo. We showed that: (1) dst1 and rpb9 mutants have a synthetic growth defective phenotype when combined with fyv4, gim5, htz1, yal011w, ybr231c, soh1, vps71, and vps72 mutants that is exacerbated during germination or at high salt concentrations; (2) TFIIS and Rpb9 are essential when the cells are challenged with microtubule-destabilizing drugs; (3) among the SDO (synthetic with Dst one), SOH1 shows the strongest genetic interaction with DST1; (4) the presence of multiple copies of TAF14, SUA7, GAL11, RTS1, and TYS1 alleviate the growth phenotype of dst1 soh1 mutants; and (5) SRB5 and SIN4 genetically interact with DST1. We propose that TFIIS is required under stress conditions and that TFIIS is important for the transition between initiation and elongation in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Benomilo/farmacologia , Proteínas de Ligação a DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Deleção de Genes , Genes Fúngicos , Testes de Sensibilidade Microbiana , Nocodazol/farmacologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/genética , Sais/farmacologia , Tiabendazol/farmacologiaRESUMO
The DNA synthesis associated with recombinational repair of chromosomal double-strand breaks (DSBs) has a lower fidelity than normal replicative DNA synthesis. Here, we use an inverted-repeat substrate to monitor the fidelity of repair of a site-specific DSB. DSB induction made by the HO endonuclease stimulates recombination >5000-fold and is associated with a >1000-fold increase in mutagenesis of an adjacent gene. We demonstrate that most break-repair-induced mutations (BRIMs) are point mutations and have a higher proportion of frameshifts than do spontaneous mutations of the same substrate. Although the REV3 translesion DNA polymerase is not required for recombination, it introduces approximately 75% of the BRIMs and approximately 90% of the base substitution mutations. Recombinational repair of the DSB is strongly dependent upon genes of the RAD52 epistasis group; however, the residual recombinants present in rad57 mutants are associated with a 5- to 20-fold increase in BRIMs. The spectrum of mutations in rad57 mutants is similar to that seen in the wild-type strain and is similarly affected by REV3. We also find that REV3 is required for the repair of MMS-induced lesions when recombinational repair is compromised. Our data suggest that Rad55p/Rad57p help limit the generation of substrates that require pol zeta during recombination.
Assuntos
DNA Polimerase Dirigida por DNA/fisiologia , Proteínas Fúngicas/fisiologia , Mutação/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases , Southern Blotting , Reparo do DNA/fisiologia , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/fisiologia , Mutação Puntual , Rad51 Recombinase , Proteína Rad52 de Recombinação e Reparo de DNARESUMO
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.
Assuntos
Senescência Celular/genética , Chaperonas Moleculares/metabolismo , Agregação Patológica de Proteínas/metabolismo , Estresse Fisiológico , Transcrição Gênica , Linhagem Celular , Sobrevivência Celular/genética , Proteínas de Choque Térmico/metabolismo , Mutação , RNA Polimerase II/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
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.
Assuntos
RNA Polimerase II/metabolismo , RNA Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Sequência de Bases , DNA Fúngico/química , DNA Fúngico/genética , Cinética , Modelos Genéticos , Mutação , Transporte Proteico , RNA Polimerase II/genética , RNA Fúngico/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
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.
Assuntos
Proteínas Fúngicas/metabolismo , Nucleotídeos/metabolismo , RNA Polimerase II/metabolismo , Transcrição Gênica , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Citidina Trifosfato/metabolismo , Proteínas Fúngicas/genética , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Polimerase II/química , RNA Polimerase II/genética , Fatores de Tempo , Uridina Trifosfato/metabolismoRESUMO
Two new studies provide in vivo ( [this issue of Molecular Cell]) and in vitro ( [this issue of Molecular Cell]) evidence that poleta functions to extend 3' strands exchanged during homologous recombination and raise the issue of how TLS polymerases are selected onto different substrates.
Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA/metabolismo , Recombinação Genética , Animais , DNA/genética , Reparo do DNA , Humanos , Modelos Genéticos , Rad51 Recombinase/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Selective gene amplification is associated with normal development, neoplasia, and drug resistance. One class of amplification events results in large arrays of inverted repeats that are often complex in structure, thus providing little information about their genesis. We made a recombination substrate in Saccharomyces cerevisiae that frequently generates palindromic duplications to repair a site-specific double-strand break in strains deleted for the SAE2 gene. The resulting palindromes are stable in sae2Delta cells, but unstable in wild-type cells. We previously proposed that the palindromes are formed by invasion and break-induced replication, followed by an unknown end joining mechanism. Here we demonstrate that palindrome formation can occur in the absence of RAD50, YKU70, and LIG4, indicating that palindrome formation defines a new class of nonhomologous end joining events. Sequence data from 24 independent palindromic duplication junctions suggest that the duplication mechanism utilizes extremely short (4-6 bp), closely spaced (2-9 bp), inverted repeats to prime DNA synthesis via an intramolecular foldback of a 3' end. In view of our data, we present a foldback priming model for how a single copy sequence is duplicated to generate a palindrome.
Assuntos
Amplificação de Genes , Genes Fúngicos , Saccharomyces cerevisiae/genética , Sequência de Bases , Southern Blotting , DNA Fúngico , Dados de Sequência MolecularRESUMO
Detection of drug-resistant variants is important for the clinical management of human immunodeficiency virus type 1 (HIV-1) infection and for studies on the evolution of drug resistance. Here we show that hybrid elements composed of the Saccharomyces cerevisiae retrotransposon Ty1 and the reverse transcriptase (RT) of HIV-1 are useful tools for detecting, monitoring, and isolating drug-resistant reverse transcriptases. This sensitive phenotypic assay is able to detect nonnucleoside reverse transcriptase inhibitor-resistant RT domains derived from mixtures of infectious molecular clones of HIV-1 in plasma and from clinical samples when the variants comprise as little as 0.3 to 1% of the virus population. Our assay can characterize the activities and drug susceptibilities of both known and novel reverse transcriptase variants and should prove useful in studies of the evolution and clinical significance of minor drug-resistant viral variants.