Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 9 de 9
Filtrar
Mais filtros

Base de dados
Tipo de documento
Intervalo de ano de publicação
1.
PLoS Genet ; 13(10): e1007041, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29036220

RESUMO

A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). We find that yeast cells with the orc4Y232C allele have a prolonged S-phase, due to compromised replication initiation at the ribosomal DNA (rDNA) locus located on chromosome XII. The inability to initiate replication at the rDNA locus results in chromosome breakage and a severely reduced rDNA copy number in the survivors, presumably helping to ensure complete replication of chromosome XII. Although reducing rDNA copy number may help ensure complete chromosome replication, orc4Y232C cells struggle to meet the high demand for ribosomal RNA synthesis. This finding provides additional evidence linking two essential cellular pathways-DNA replication and ribosome biogenesis.


Assuntos
Proteínas de Ciclo Celular/genética , Microtia Congênita/genética , Replicação do DNA/genética , Transtornos do Crescimento/genética , Micrognatismo/genética , Complexo de Reconhecimento de Origem/genética , Patela/anormalidades , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Aminoácidos/genética , Quebra Cromossômica , DNA Ribossômico/genética , Humanos , Mutação de Sentido Incorreto , Patela/fisiologia , RNA Ribossômico , Saccharomyces cerevisiae/genética
2.
PLoS Genet ; 8(5): e1002677, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22589733

RESUMO

The centromeric regions of all Saccharomyces cerevisiae chromosomes are found in early replicating domains, a property conserved among centromeres in fungi and some higher eukaryotes. Surprisingly, little is known about the biological significance or the mechanism of early centromere replication; however, the extensive conservation suggests that it is important for chromosome maintenance. Do centromeres ensure their early replication by promoting early activation of nearby origins, or have they migrated over evolutionary time to reside in early replicating regions? In Candida albicans, a neocentromere contains an early firing origin, supporting the first hypothesis but not addressing whether the new origin is intrinsically early firing or whether the centromere influences replication time. Because the activation time of individual origins is not an intrinsic property of S. cerevisiae origins, but is influenced by surrounding sequences, we sought to test the hypothesis that centromeres influence replication time by moving a centromere to a late replication domain. We used a modified Meselson-Stahl density transfer assay to measure the kinetics of replication for regions of chromosome XIV in which either the functional centromere or a point-mutated version had been moved near origins that reside in a late replication region. We show that a functional centromere acts in cis over a distance as great as 19 kb to advance the initiation time of origins. Our results constitute a direct link between establishment of the kinetochore and the replication initiation machinery, and suggest that the proposed higher-order structure of the pericentric chromatin influences replication initiation.


Assuntos
Centrômero/genética , Replicação do DNA , Cinetocoros , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Cromatina/genética , Cromossomos/genética , Cromossomos Fúngicos/genética , Fase S/genética
3.
MAbs ; 16(1): 2394230, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-39192463

RESUMO

We previously described an in vitro single-chain fragment (scFv) library platform originally designed to generate antibodies with excellent developability properties. The platform design was based on the use of clinical antibodies as scaffolds into which replicated natural complementarity-determining regions purged of sequence liabilities were inserted, and the use of phage and yeast display to carry out antibody selection. In addition to being developable, antibodies generated using our platform were extremely diverse, with most campaigns yielding sub-nanomolar binders. Here, we describe a platform advancement that incorporates Fab phage display followed by single-chain antibody-binding fragment Fab (scFab) yeast display. The scFab single-gene format provides balanced expression of light and heavy chains, with enhanced conversion to IgG, thereby combining the advantages of scFvs and Fabs. A meticulously engineered, quality-controlled Fab phage library was created using design principles similar to those used to create the scFv library. A diverse panel of binding scFabs, with high conversion efficiency to IgG, was isolated against two targets. This study highlights the compatibility of phage and yeast display with a Fab semi-synthetic library design, offering an efficient approach to generate drug-like antibodies directly, facilitating their conversion to potential therapeutic candidates.


Assuntos
Afinidade de Anticorpos , Fragmentos Fab das Imunoglobulinas , Biblioteca de Peptídeos , Anticorpos de Cadeia Única , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/química , Humanos , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/química , Afinidade de Anticorpos/imunologia , Técnicas de Visualização da Superfície Celular/métodos , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/química
4.
DNA Repair (Amst) ; 84: 102633, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31231063

RESUMO

An R-loop is a structure that forms when an RNA transcript stays bound to the DNA strand that encodes it and leaves the complementary strand exposed as a loop of single stranded DNA. R-loops accumulate when the processing of RNA transcripts is impaired. The failure to remove these RNA-DNA hybrids can lead to replication fork stalling and genome instability. Resolution of R-loops is thought to be mediated mainly by RNase H enzymes through the removal and degradation of the RNA in the hybrid. However, DNA helicases can also dismantle R-loops by displacing the bound RNA. In particular, the Pif1 family DNA helicases have been shown to regulate R-loop formation at specific genomic loci, such as tRNA genes and centromeres. Here we review the roles of Pif1 family helicases in vivo and in vitro and discuss evidence that Pif1 family helicases act on RNA-DNA hybrids and highlight their potential roles in complementing RNase H for R-loop resolution.


Assuntos
Estruturas R-Loop , Ribonuclease H/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
5.
Genetics ; 213(2): 465-479, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31391265

RESUMO

Two common features of centromeres are their transcription into noncoding centromere RNAs (cen-RNAs) and their assembly into nucleosomes that contain a centromere-specific histone H3 (cenH3). Here, we show that Saccharomyces cerevisiae cen-RNA was present in low amounts in wild-type (WT) cells, and that its appearance was tightly cell cycle-regulated, appearing and disappearing in a narrow window in S phase after centromere replication. In cells lacking Cbf1, a centromere-binding protein, cen-RNA was 5-12 times more abundant throughout the cell cycle. In WT cells, cen-RNA appearance occurred at the same time as loss of Cbf1's centromere binding, arguing that the physical presence of Cbf1 inhibits cen-RNA production. Binding of the Pif1 DNA helicase, which happens in mid-late S phase, occurred at about the same time as Cbf1 loss from the centromere, suggesting that Pif1 may facilitate this loss by its known ability to displace proteins from DNA. Cen-RNAs were more abundant in rnh1Δ cells but only in mid-late S phase. However, fork pausing at centromeres was not elevated in rnh1Δ cells but rather was due to centromere-binding proteins, including Cbf1 Strains with increased cen-RNA lost centromere plasmids at elevated rates. In cbf1Δ cells, where both the levels and the cell cycle-regulated appearance of cen-RNA were disrupted, the timing and levels of cenH3 centromere binding were perturbed. Thus, cen-RNAs are highly regulated, and disruption of this regulation correlates with changes in centromere structure and function.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Centrômero/genética , DNA Helicases/genética , Histonas/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromatina/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Cinetocoros , Nucleossomos/genética , RNA Fúngico/genética , RNA não Traduzido/genética , Saccharomyces cerevisiae/genética
6.
Genetics ; 211(1): 105-119, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30442759

RESUMO

Pif1 family helicases are found in virtually all eukaryotes. Saccharomyces cerevisiae (Sc) encodes two Pif1 family helicases, ScPif1 and Rrm3 ScPif1 is multifunctional, required not only for maintenance of mitochondrial DNA but also for multiple distinct nuclear functions. Rrm3 moves with the replication fork and promotes movement of the fork through ∼1400 hard-to-replicate sites, including centromeres. Here we show that ScPif1, like Rrm3, bound robustly to yeast centromeres but only if the centromere was active. While Rrm3 binding to centromeres occurred in early to mid S phase, about the same time as centromere replication, ScPif1 binding occurred later in the cell cycle when replication of most centromeres is complete. However, the timing of Rrm3 and ScPif1 centromere binding was altered by the absence of the other helicase, such that Rrm3 centromere binding occurred later in pif1-m2 cells and ScPif1 centromere binding occurred earlier in rrm3Δ cells. As shown previously, the modest pausing of replication forks at centromeres seen in wild-type cells was increased in the absence of Rrm3 While a lack of ScPif1 did not result in increased fork pausing at centromeres, pausing was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Likewise, centromere function as monitored by the loss rate of a centromere plasmid was increased in rrm3Δ but not pif1Δ cells, and was even higher in rrm3Δ pif1Δ cells than in rrm3Δ cells. Thus, ScPif1 promotes centromere replication and segregation, but only in the absence of Rrm3 These data also hint at a potential post-S phase function for ScPif1 at centromeres. These studies add to the growing list of ScPif1 functions that promote chromosome stability.


Assuntos
Segregação de Cromossomos , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Saccharomyces cerevisiae/metabolismo , Centrômero/genética , DNA Helicases/genética , Farmacorresistência Fúngica/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Moduladores de Tubulina/toxicidade
7.
Nat Commun ; 8: 15025, 2017 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-28429714

RESUMO

Saccharomyces cerevisiae encodes two Pif1 family DNA helicases, Pif1 and Rrm3. Rrm3 promotes DNA replication past stable protein complexes at tRNA genes (tDNAs). We identify a new role for the Pif1 helicase: promotion of replication and suppression of DNA damage at tDNAs. Pif1 binds multiple tDNAs, and this binding is higher in rrm3Δ cells. Accumulation of replication intermediates and DNA damage at tDNAs is higher in pif1Δ rrm3Δ than in rrm3Δ cells. DNA damage at tDNAs in the absence of these helicases is suppressed by destabilizing R-loops while Pif1 and Rrm3 binding to tDNAs is increased upon R-loop stabilization. We propose that Rrm3 and Pif1 promote genome stability at tDNAs by displacing the stable multi-protein transcription complex and by removing R-loops. Thus, we identify tDNAs as a new source of R-loop-mediated DNA damage. Given their large number and high transcription rate, tDNAs may be a potent source of genome instability.


Assuntos
DNA Helicases/genética , DNA Fúngico/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , DNA Fúngico/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Ligação Proteica , RNA de Transferência/genética , RNA de Transferência/metabolismo , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
8.
G3 (Bethesda) ; 3(11): 1955-63, 2013 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-24022751

RESUMO

Eukaryotic origins of DNA replication undergo activation at various times in S-phase, allowing the genome to be duplicated in a temporally staggered fashion. In the budding yeast Saccharomyces cerevisiae, the activation times of individual origins are not intrinsic to those origins but are instead governed by surrounding sequences. Currently, there are two examples of DNA sequences that are known to advance origin activation time, centromeres and forkhead transcription factor binding sites. By combining deletion and linker scanning mutational analysis with two-dimensional gel electrophoresis to measure fork direction in the context of a two-origin plasmid, we have identified and characterized a 19- to 23-bp and a larger 584-bp DNA sequence that are capable of advancing origin activation time.


Assuntos
DNA/metabolismo , Origem de Replicação/genética , Saccharomyces cerevisiae/genética , Sequência de Bases , Sítios de Ligação , Centrômero/genética , Centrômero/metabolismo , DNA/química , Análise Mutacional de DNA , Proteínas de Ligação a DNA/genética , Eletroforese em Gel Bidimensional , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Genoma Fúngico , Dados de Sequência Molecular , Mutagênese , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
9.
Mol Cell Biol ; 28(3): 897-906, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18039855

RESUMO

Homologous recombination (HR) is critical for DNA double-strand break (DSB) repair and genome stabilization. In yeast, HR is catalyzed by the Rad51 strand transferase and its "mediators," including the Rad52 single-strand DNA-annealing protein, two Rad51 paralogs (Rad55 and Rad57), and Rad54. A Rad51 homolog, Dmc1, is important for meiotic HR. In wild-type cells, most DSB repair results in gene conversion, a conservative HR outcome. Because Rad51 plays a central role in the homology search and strand invasion steps, DSBs either are not repaired or are repaired by nonconservative single-strand annealing or break-induced replication mechanisms in rad51Delta mutants. Although DSB repair by gene conversion in the absence of Rad51 has been reported for ectopic HR events (e.g., inverted repeats or between plasmids), Rad51 has been thought to be essential for DSB repair by conservative interchromosomal (allelic) gene conversion. Here, we demonstrate that DSBs stimulate gene conversion between homologous chromosomes (allelic conversion) by >30-fold in a rad51Delta mutant. We show that Rad51-independent allelic conversion and break-induced replication occur independently of Rad55, Rad57, and Dmc1 but require Rad52. Unlike DSB-induced events, spontaneous allelic conversion was detected in both rad51Delta and rad52Delta mutants, but not in a rad51Delta rad52Delta double mutant. The frequencies of crossovers associated with DSB-induced gene conversion were similar in the wild type and the rad51Delta mutant, but discontinuous conversion tracts were fivefold more frequent and tract lengths were more widely distributed in the rad51Delta mutant, indicating that heteroduplex DNA has an altered structure, or is processed differently, in the absence of Rad51.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases , Enzimas Reparadoras do DNA , Conversão Gênica , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA