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1.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
2.
Nat Commun ; 12(1): 2111, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833229

RESUMO

Smc5/6 is essential for genome structural integrity by yet unknown mechanisms. Here we find that Smc5/6 co-localizes with the DNA crossed-strand processing complex Sgs1-Top3-Rmi1 (STR) at genomic regions known as natural pausing sites (NPSs) where it facilitates Top3 retention. Individual depletions of STR subunits and Smc5/6 cause similar accumulation of joint molecules (JMs) composed of reversed forks, double Holliday Junctions and hemicatenanes, indicative of Smc5/6 regulating Sgs1 and Top3 DNA processing activities. We isolate an intra-allelic suppressor of smc6-56 proficient in Top3 retention but affected in pathways that act complementarily with Sgs1 and Top3 to resolve JMs arising at replication termination. Upon replication stress, the smc6-56 suppressor requires STR and Mus81-Mms4 functions for recovery, but not Srs2 and Mph1 helicases that prevent maturation of recombination intermediates. Thus, Smc5/6 functions jointly with Top3 and STR to mediate replication completion and influences the function of other DNA crossed-strand processing enzymes at NPSs.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA/genética , Genoma Fúngico/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo
3.
Curr Opin Genet Dev ; 71: 27-33, 2021 12.
Artigo em Inglês | MEDLINE | ID: mdl-34271541

RESUMO

Helicases are in the spotlight of DNA metabolism and are critical for DNA repair in all domains of life. At their biochemical core, they bind and hydrolyze ATP, converting this energy to translocate unidirectionally, with different strand polarities and substrate binding specificities, along one strand of a nucleic acid. In doing so, DNA and RNA helicases separate duplex strands or remove nucleoprotein complexes, affecting DNA repair and the architecture of replication forks. In this review, we focus on recent advances on the roles and regulations of DNA helicases in homologous recombination repair, a critical pathway for mending damaged chromosomes and for ensuring genome integrity.


Assuntos
DNA Helicases , Reparo de DNA por Recombinação , Dano ao DNA , DNA Helicases/genética , Reparo do DNA/genética , Replicação do DNA/genética , Recombinação Homóloga
4.
Nat Commun ; 11(1): 5746, 2020 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-33184279

RESUMO

The Mus81-Mms4 nuclease is activated in G2/M via Mms4 phosphorylation to allow resolution of persistent recombination structures. However, the fate of the activated phosphorylated Mms4 remains unknown. Here we find that Mms4 is engaged by (poly)SUMOylation and ubiquitylation and targeted for proteasome degradation, a process linked to the previously described Mms4 phosphorylation cycle. Mms4 is a mitotic substrate for the SUMO-Targeted Ubiquitin ligase Slx5/8, the SUMO-like domain-containing protein Esc2, and the Mms1-Cul8 ubiquitin ligase. In the absence of these activities, phosphorylated Mms4 accumulates on chromatin in an active state in the next G1, subsequently causing abnormal processing of replication-associated recombination intermediates and delaying the activation of the DNA damage checkpoint. Mus81-Mms4 mutants that stabilize phosphorylated Mms4 have similar detrimental effects on genome integrity. Overall, our findings highlight a replication protection function for Esc2-STUbL-Cul8 and emphasize the importance for genome stability of resetting phosphorylated Mms4 from one cycle to another.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Mitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Proteínas Culina/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Endonucleases Flap/genética , Regulação Fúngica da Expressão Gênica , Instabilidade Genômica , Mitose/genética , Processamento de Proteína Pós-Traducional/genética , Processamento de Proteína Pós-Traducional/fisiologia , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
5.
Sci Adv ; 6(15): eaaz3327, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32285001

RESUMO

DNA damage tolerance (DDT) is crucial for genome integrity maintenance. DDT is mainly carried out by template switch recombination, an error-free mode of overcoming DNA lesions, or translesion DNA synthesis, which is error-prone. Here, we investigated the role of Mgs1/WRNIP1 in modulating DDT. Using budding yeast, we found that elimination of Mgs1 in cells lacking Rad5, an essential protein for DDT, activates an alternative mode of DNA damage bypass, driven by recombination, which allows chromosome replication and cell viability under stress conditions that block DNA replication forks. This salvage pathway is RAD52 and RAD59 dependent, requires the DNA polymerase δ and PCNA modification at K164, and is enabled by Esc2 and the PCNA unloader Elg1, being inhibited when Mgs1 is present. We propose that Mgs1 is necessary to prevent a potentially toxic recombination salvage pathway at sites of perturbed replication, which, in turn, favors Rad5-dependent template switching, thus helping to preserve genome stability.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , Recombinação Genética , Transdução de Sinais , DNA Helicases/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Instabilidade Genômica , Viabilidade Microbiana/genética , Modelos Biológicos , Saccharomycetales/genética , Saccharomycetales/metabolismo , Estresse Fisiológico
6.
Mol Cell ; 73(5): 900-914.e9, 2019 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-30733119

RESUMO

Post-replication repair (PRR) allows tolerance of chemical- and UV-induced DNA base lesions in both an error-free and an error-prone manner. In classical PRR, PCNA monoubiquitination recruits translesion synthesis (TLS) DNA polymerases that can replicate through lesions. We find that PRR responds to DNA replication stress that does not cause base lesions. Rad5 forms nuclear foci during normal S phase and after exposure to types of replication stress where DNA base lesions are likely absent. Rad5 binds to the sites of stressed DNA replication forks, where it recruits TLS polymerases to repair single-stranded DNA (ssDNA) gaps, preventing mitotic defects and chromosome breaks. In contrast to the prevailing view of PRR, our data indicate that Rad5 promotes both mutagenic and error-free repair of undamaged ssDNA that arises during physiological and exogenous replication stress.


Assuntos
Quebras de DNA de Cadeia Simples , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , DNA Fúngico/metabolismo , DNA de Cadeia Simples/metabolismo , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sítios de Ligação , Cromossomos Fúngicos , DNA Helicases/genética , DNA Fúngico/genética , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mitose , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitinação
7.
Nat Commun ; 9(1): 3680, 2018 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-30206225

RESUMO

Genome instability is associated with tumorigenesis. Here, we identify a role for the histone Htz1, which is deposited by the Swr1 chromatin-remodeling complex (SWR-C), in preventing genome instability in the absence of the replication fork/replication checkpoint proteins Mrc1, Csm3, or Tof1. When combined with deletion of SWR1 or HTZ1, deletion of MRC1, CSM3, or TOF1 or a replication-defective mrc1 mutation causes synergistic increases in gross chromosomal rearrangement (GCR) rates, accumulation of a broad spectrum of GCRs, and hypersensitivity to replication stress. The double mutants have severe replication defects and accumulate aberrant replication intermediates. None of the individual mutations cause large increases in GCR rates; however, defects in MRC1, CSM3 or TOF1 cause activation of the DNA damage checkpoint and replication defects. We propose a model in which Htz1 deposition and retention in chromatin prevents transiently stalled replication forks that occur in mrc1, tof1, or csm3 mutants from being converted to DNA double-strand breaks that trigger genome instability.


Assuntos
Adenosina Trifosfatases/metabolismo , Montagem e Desmontagem da Cromatina , Replicação do DNA , Instabilidade Genômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Ciclo Celular , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Cromossomos Fúngicos/genética , Dano ao DNA , Replicação do DNA/efeitos dos fármacos , Rearranjo Gênico/genética , Recombinação Homóloga/genética , Hidroxiureia/farmacologia , Modelos Biológicos , Mutação/genética , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos
8.
EMBO J ; 37(18)2018 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-30111537

RESUMO

DNA damage tolerance (DDT) mechanisms facilitate replication resumption and completion when DNA replication is blocked by bulky DNA lesions. In budding yeast, template switching (TS) via the Rad18/Rad5 pathway is a favored DDT pathway that involves usage of the sister chromatid as a template to bypass DNA lesions in an error-free recombination-like process. Here, we establish that the Snf2 family translocase Irc5 is a novel factor that promotes TS and averts single-stranded DNA persistence during replication. We demonstrate that, during replication stress, Irc5 enables replication progression by assisting enrichment of cohesin complexes, recruited in an Scc2/Scc4-dependent fashion, near blocked replication forks. This allows efficient formation of sister chromatid junctions that are crucial for error-free DNA lesion bypass. Our results support the notion of a key role of cohesin in the completion of DNA synthesis under replication stress and reveal that the Rad18/Rad5-mediated DDT pathway is linked to cohesin enrichment at sites of perturbed replication via the Snf2 family translocase Irc5.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Replicação do DNA , DNA Fúngico/biossíntese , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Sistema Livre de Células/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , DNA Helicases , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
9.
Nucleic Acids Res ; 46(15): 7586-7611, 2018 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-30011030

RESUMO

The Saccharomyces cerevisiae kinase/adenosine triphosphatase Rio1 regulates rDNA transcription and segregation, pre-rRNA processing and small ribosomal subunit maturation. Other roles are unknown. When overexpressed, human ortholog RIOK1 drives tumor growth and metastasis. Likewise, RIOK1 promotes 40S ribosomal subunit biogenesis and has not been characterized globally. We show that Rio1 manages directly and via a series of regulators, an essential signaling network at the protein, chromatin and RNA levels. Rio1 orchestrates growth and division depending on resource availability, in parallel to the nutrient-activated Tor1 kinase. To define the Rio1 network, we identified its physical interactors, profiled its target genes/transcripts, mapped its chromatin-binding sites and integrated our data with yeast's protein-protein and protein-DNA interaction catalogs using network computation. We experimentally confirmed network components and localized Rio1 also to mitochondria and vacuoles. Via its network, Rio1 commands protein synthesis (ribosomal gene expression, assembly and activity) and turnover (26S proteasome expression), and impinges on metabolic, energy-production and cell-cycle programs. We find that Rio1 activity is conserved to humans and propose that pathological RIOK1 may fuel promiscuous transcription, ribosome production, chromosomal instability, unrestrained metabolism and proliferation; established contributors to cancer. Our study will advance the understanding of numerous processes, here revealed to depend on Rio1 activity.


Assuntos
Ciclo Celular/genética , Metabolismo Energético/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromatina/metabolismo , Segregação de Cromossomos/genética , Mitocôndrias/genética , Fosfatidilinositol 3-Quinases/metabolismo , RNA Fúngico/genética , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Transcrição Genética/genética
10.
Crit Rev Biochem Mol Biol ; 52(4): 381-394, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28325102

RESUMO

The complete and faithful duplication of the genome is an essential prerequisite for proliferating cells to maintain genome integrity. This objective is greatly challenged by DNA damage encountered during replication, which causes fork stalling and in certain cases, fork breakage. DNA damage tolerance (DDT) pathways mitigate the effects on fork stability induced by replication fork stalling by mediating damage-bypass and replication fork restart. These DDT mechanisms, largely relying on homologous recombination (HR) and specialized polymerases, can however contribute to genome rearrangements and mutagenesis. There is a profound connection between replication and recombination: recombination proteins protect replication forks from nuclease-mediated degradation of the nascent DNA strands and facilitate replication completion in cells challenged by DNA damage. Moreover, in case of fork collapse and formation of double strand breaks (DSBs), the recombination factors present or recruited to the fork facilitate HR-mediated DSB repair, which is primarily error-free. Disruption of HR is inexorably linked to genome instability, but the premature activation of HR during replication often leads to genome rearrangements. Faithful replication necessitates the downregulation of HR and disruption of active RAD51 filaments at replication forks, but upon persistent fork stalling, building up of HR is critical for the reorganization of the replication fork and for filling-in of the gaps associated with discontinuous replication induced by DNA lesions. Here we summarize and reflect on our understanding of the mechanisms that either suppress recombination or locally enhance it during replication, and the principles that underlie this regulation.


Assuntos
Replicação do DNA , Recombinação Genética , Animais , Dano ao DNA , Reparo do DNA , Humanos
11.
Nucleic Acids Res ; 45(1): 215-230, 2017 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-27694623

RESUMO

Replication across damaged DNA templates is accompanied by transient formation of sister chromatid junctions (SCJs). Cells lacking Esc2, an adaptor protein containing no known enzymatic domains, are defective in the metabolism of these SCJs. However, how Esc2 is involved in the metabolism of SCJs remains elusive. Here we show interaction between Esc2 and a structure-specific endonuclease Mus81-Mms4 (the Mus81 complex), their involvement in the metabolism of SCJs, and the effects Esc2 has on the enzymatic activity of the Mus81 complex. We found that Esc2 specifically interacts with the Mus81 complex via its SUMO-like domains, stimulates enzymatic activity of the Mus81 complex in vitro, and is involved in the Mus81 complex-dependent resolution of SCJs in vivo Collectively, our data point to the possibility that the involvement of Esc2 in the metabolism of SCJs is, in part, via modulation of the activity of the Mus81 complex.


Assuntos
Cromátides/metabolismo , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular , Cromátides/química , Clonagem Molecular , Dano ao DNA , Replicação do DNA , DNA Cruciforme/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Endonucleases/química , Endonucleases/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Instabilidade Genômica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/química , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
12.
Cell Rep ; 16(2): 368-378, 2016 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-27373152

RESUMO

Timely removal of DNA recombination intermediates is critical for genome stability. The DNA helicase-topoisomerase complex, Sgs1-Top3-Rmi1 (STR), is the major pathway for processing these intermediates to generate conservative products. However, the mechanisms that promote STR-mediated functions remain to be defined. Here we show that Sgs1 binds to poly-SUMO chains and associates with the Smc5/6 SUMO E3 complex in yeast. Moreover, these interactions contribute to the sumoylation of Sgs1, Top3, and Rmi1 upon the generation of recombination structures. We show that reduced STR sumoylation leads to accumulation of recombination structures, and impaired growth in conditions when these structures arise frequently, highlighting the importance of STR sumoylation. Mechanistically, sumoylation promotes STR inter-subunit interactions and accumulation at DNA repair centers. These findings expand the roles of sumoylation and Smc5/6 in genome maintenance by demonstrating that they foster STR functions in the removal of recombination intermediates.


Assuntos
Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sumoilação , Sequência de Aminoácidos , Proteínas de Ciclo Celular/fisiologia , Replicação do DNA , DNA Fúngico/genética , DNA Fúngico/metabolismo , Recombinação Genética , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/fisiologia , Técnicas do Sistema de Duplo-Híbrido
13.
DNA Repair (Amst) ; 44: 68-75, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27236213

RESUMO

Replication perturbations activate DNA damage tolerance (DDT) pathways, which are crucial to promote replication completion and to prevent fork breakage, a leading cause of genome instability. One mode of DDT uses translesion synthesis polymerases, which however can also introduce mutations. The other DDT mode involves recombination-mediated mechanisms, which are generally accurate. DDT occurs prevalently postreplicatively, but in certain situations homologous recombination is needed to restart forks. Fork reversal can function to stabilize stalled forks, but may also promote error-prone outcome when used for fork restart. Recent years have witnessed important advances in our understanding of the mechanisms and DNA structures that mediate recombination-mediated damage-bypass and highlighted principles that regulate DDT pathway choice locally and temporally. In this review we summarize the current knowledge and paradoxes on recombination-mediated DDT pathways and their workings, discuss how the intermediate DNA structures may influence genome integrity, and outline key open questions for future research.


Assuntos
Reparo de Erro de Pareamento de DNA , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Animais , Pareamento Incorreto de Bases , DNA/química , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Instabilidade Genômica , Humanos , Mamíferos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Nucleus ; 7(1): 8-12, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26889705

RESUMO

Genome duplication is coupled with DNA damage tolerance (DDT) and chromatin structural changes. Recently we reported that mutations in Primase subunits or factors that bridge Polα/Primase with the replicative helicase, Ctf4, caused abnormal usage of DDT pathways, negatively influenced sister chromatid cohesion (SCC), and associated with increased fork reversal. (1) We also found that cohesin, which is paradigmatic for SCC, facilitates recombination-mediated DDT. However, only the recombination defects of cohesin, but not of cohesion-defective Polα/Primase/Ctf4 mutants, were rescued by artificial tethering of sister chromatids. Genetic tests and electron microscopy analysis of replication intermediates made us propose that management of single-stranded DNA forming proximal to the fork is a critical determinant of chromosome and replication fork structure, and influences DDT pathway choice. Here we discuss the implications of our findings for understanding DDT regulation and cohesion establishment during replication, and outline directions to rationalize the relationship between these chromosome metabolism processes.


Assuntos
DNA Polimerase I/metabolismo , DNA Primase/metabolismo , Replicação do DNA/fisiologia , DNA Fúngico/biossíntese , Proteínas de Ligação a DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Polimerase I/genética , DNA Primase/genética , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
15.
J Biol Chem ; 291(9): 4442-52, 2016 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-26740628

RESUMO

DNA damage must be repaired in an accurate and timely fashion to preserve genome stability. Cellular mechanisms preventing genome instability are crucial to human health because genome instability is considered a hallmark of cancer. Collectively referred to as the DNA damage response, conserved pathways ensure proper DNA damage recognition and repair. The function of numerous DNA damage response components is fine-tuned by posttranslational modifications, including ubiquitination. This not only involves the enzyme cascade responsible for conjugating ubiquitin to substrates but also requires enzymes that mediate directed removal of ubiquitin. Deubiquitinases remove ubiquitin from substrates to prevent degradation or to mediate signaling functions. The Saccharomyces cerevisiae deubiquitinase Ubp7 has been characterized previously as an endocytic factor. However, here we identify Ubp7 as a novel factor affecting S phase progression after hydroxyurea treatment and demonstrate an evolutionary and genetic interaction of Ubp7 with DNA damage repair pathways of homologous recombination and nucleotide excision repair. We find that deletion of UBP7 sensitizes cells to hydroxyurea and cisplatin and demonstrate that factors that stabilize replication forks are critical under these conditions. Furthermore, ubp7Δ cells exhibit an S phase progression defect upon checkpoint activation by hydroxyurea treatment. ubp7Δ mutants are epistatic to factors involved in histone maintenance and modification, and we find that a subset of Ubp7 is chromatin-associated. In summary, our results suggest that Ubp7 contributes to S phase progression by affecting the chromatin state at replication forks, and we propose histone H2B ubiquitination as a potential substrate of Ubp7.


Assuntos
Cromatina/enzimologia , Proteínas Fúngicas/metabolismo , Fase S , Saccharomycetales/enzimologia , Proteases Específicas de Ubiquitina/metabolismo , Cromatina/efeitos dos fármacos , Cromatina/metabolismo , Cisplatino/farmacologia , Reagentes para Ligações Cruzadas/farmacologia , Reparo do DNA , Replicação do DNA/efeitos dos fármacos , Proteínas Fúngicas/genética , Deleção de Genes , Instabilidade Genômica/efeitos dos fármacos , Histonas/metabolismo , Hidroxiureia/farmacologia , Viabilidade Microbiana/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico/farmacologia , Fase S/efeitos dos fármacos , Saccharomycetales/citologia , Saccharomycetales/efeitos dos fármacos , Saccharomycetales/crescimento & desenvolvimento , Proteases Específicas de Ubiquitina/genética
17.
Mol Cell ; 60(2): 268-79, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26439300

RESUMO

Elucidating the individual and collaborative functions of genome maintenance factors is critical for understanding how genome duplication is achieved. Here, we investigate a conserved scaffold in budding yeast, Rtt107, and its three partners: a SUMO E3 complex, a ubiquitin E3 complex, and Slx4. Biochemical and genetic findings show that Rtt107 interacts separately with these partners and contributes to their individual functions, including a role in replisome sumoylation. We also provide evidence that Rtt107 associates with replisome components, and both itself and its associated E3s are important for replicating regions far from initiation sites. Corroborating these results, replication defects due to Rtt107 loss and genotoxic sensitivities in mutants of Rtt107 and its associated E3s are rescued by increasing replication initiation events through mutating two master repressors of late origins, Mrc1 and Mec1. These findings suggest that Rtt107 functions as a multi-functional platform to support replication progression with its partner E3 enzymes.


Assuntos
Replicação do DNA , Endodesoxirribonucleases/genética , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteína SUMO-1/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Endodesoxirribonucleases/metabolismo , Genoma Fúngico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , /metabolismo , Proteína SUMO-1/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sumoilação , Ubiquitina-Proteína Ligases/metabolismo
18.
Genes Dev ; 29(19): 2067-80, 2015 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-26443850

RESUMO

Accurate completion of replication relies on the ability of cells to activate error-free recombination-mediated DNA damage bypass at sites of perturbed replication. However, as anti-recombinase activities are also recruited to replication forks, how recombination-mediated damage bypass is enabled at replication stress sites remained puzzling. Here we uncovered that the conserved SUMO-like domain-containing Saccharomyces cerevisiae protein Esc2 facilitates recombination-mediated DNA damage tolerance by allowing optimal recruitment of the Rad51 recombinase specifically at sites of perturbed replication. Mechanistically, Esc2 binds stalled replication forks and counteracts the anti-recombinase Srs2 helicase via a two-faceted mechanism involving chromatin recruitment and turnover of Srs2. Importantly, point mutations in the SUMO-like domains of Esc2 that reduce its interaction with Srs2 cause suboptimal levels of Rad51 recruitment at damaged replication forks. In conclusion, our results reveal how recombination-mediated DNA damage tolerance is locally enabled at sites of replication stress and globally prevented at undamaged replicating chromosomes.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Proteínas Nucleares/metabolismo , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular , Cromatina/metabolismo , Dano ao DNA/genética , DNA Helicases/metabolismo , Proteínas Nucleares/genética , Mutação Puntual , Ligação Proteica , Rad51 Recombinase/metabolismo
19.
Genes Dev ; 29(10): 1000-5, 2015 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-25956905

RESUMO

Budding yeast Mph1 helicase and its orthologs drive multiple DNA transactions. Elucidating the mechanisms that regulate these motor proteins is central to understanding genome maintenance processes. Here, we show that the conserved histone fold MHF complex promotes Mph1-mediated repair of damaged replication forks but does not influence the outcome of DNA double-strand break repair. Mechanistically, scMHF relieves the inhibition imposed by the structural maintenance of chromosome protein Smc5 on Mph1 activities relevant to replication-associated repair through binding to Mph1 but not DNA. Thus, scMHF is a function-specific enhancer of Mph1 that enables flexible response to different genome repair situations.


Assuntos
Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , RNA Helicases DEAD-box/metabolismo , DNA/genética , Reparo do DNA , Genoma Fúngico/genética , Mutação , Ligação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
20.
Nucleic Acids Res ; 43(5): 2666-77, 2015 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-25690888

RESUMO

Many genome maintenance factors have multiple enzymatic activities. In most cases, how their distinct activities functionally relate with each other is unclear. Here we examined the conserved budding yeast Rad5 protein that has both ubiquitin ligase and DNA helicase activities. The Rad5 ubiquitin ligase activity mediates PCNA poly-ubiquitination and subsequently recombination-based DNA lesion tolerance. Interestingly, the ligase domain is embedded in a larger helicase domain comprising seven consensus motifs. How features of the helicase domain influence ligase function is controversial. To clarify this issue, we use genetic, 2D gel and biochemical analyses and show that a Rad5 helicase motif important for ATP binding is also required for PCNA poly-ubiquitination and recombination-based lesion tolerance. We determine that this requirement is due to a previously unrecognized contribution of the motif to the PCNA and ubiquitination enzyme interaction, and not due to its canonical role in supporting helicase activity. We further show that Rad5's helicase-mediated contribution to replication stress survival is separable from recombination. These findings delineate how two Rad5 enzymatic domains concertedly influence PCNA modification, and unveil their discrete contributions to stress tolerance.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA Helicases/genética , Replicação do DNA/genética , Eletroforese em Gel Bidimensional , Immunoblotting , Mutação , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Sumoilação , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
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