RESUMO
During homologous recombination, cells must coordinate repair, DNA damage checkpoint signaling, and movement of chromosomal loci to facilitate homology search. In Saccharomyces cerevisiae, increased movement of damaged loci (local mobility) and undamaged loci (global mobility) precedes homolog pairing in mitotic cells. How cells modulate chromosome mobility in response to DNA damage remains unclear. Here, we demonstrate that global chromosome mobility is regulated by the Rad51 recombinase and its mediator, Rad52. Surprisingly, rad51Δ rad52Δ cells display checkpoint-dependent constitutively increased mobility, indicating that a regulatory circuit exists between recombination and checkpoint machineries to govern chromosomal mobility. We found that the requirement for Rad51 in this circuit is distinct from its role in recombination and that interaction with Rad52 is necessary to alleviate inhibition imposed by mediator recruitment to ssDNA. Thus, interplay between recombination factors and the checkpoint restricts increased mobility until recombination proteins are assembled at damaged sites.
Assuntos
Cromossomos Fúngicos/metabolismo , Dano ao DNA , Recombinação Homóloga , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Mutação , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Códon de Terminação , Edição de Genes/métodos , Inativação Gênica , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Códon sem Sentido , Biologia Computacional , Enzimas de Restrição do DNA/genética , Enzimas de Restrição do DNA/metabolismo , Bases de Dados Genéticas , Regulação Fúngica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Células HEK293 , Humanos , Camundongos , Neoplasias/genética , Neoplasias/metabolismo , Polimorfismo de Fragmento de Restrição , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Ratos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TransfecçãoRESUMO
The postreplication repair gene, HLTF, is often amplified and overexpressed in cancer. Here we model HLTF dysregulation through the functionally conserved Saccharomyces cerevisiae ortholog, RAD5. Genetic interaction profiling and landscape enrichment analysis of RAD5 overexpression (RAD5OE) reveals requirements for genes involved in recombination, crossover resolution, and DNA replication. While RAD5OE and rad5Δ both cause cisplatin sensitivity and share many genetic interactions, RAD5OE specifically requires crossover resolving genes and drives recombination in a region of repetitive DNA. Remarkably, RAD5OE induced recombination does not require other post-replication repair pathway members, or the PCNA modification sites involved in regulation of this pathway. Instead, the RAD5OE phenotype depends on a conserved domain necessary for binding 3' DNA ends. Analysis of DNA replication intermediates supports a model in which dysregulated Rad5 causes aberrant template switching at replication forks. The direct effect of Rad5 on replication forks in vivo, increased recombination, and cisplatin sensitivity predicts similar consequences for dysregulated HLTF in cancer.
Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Recombinação Genética/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Cisplatino/farmacologia , Troca Genética/genética , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/genética , Replicação do DNA/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/genética , Saccharomyces cerevisiae/genéticaRESUMO
During S phase in Saccharomyces cerevisiae, chromosomal loci become mobile in response to DNA double-strand breaks both at the break site (local mobility) and throughout the nucleus (global mobility). Increased nuclear exploration is regulated by the recombination machinery and the DNA damage checkpoint and is likely an important aspect of homology search. While mobility in response to DNA damage has been studied extensively in S phase, the response in interphase has not, and the question of whether homologous recombination proceeds to completion in G1 phase remains controversial. Here, we find that global mobility is triggered in G1 phase. As in S phase, global mobility in G1 phase is controlled by the DNA damage checkpoint and the Rad51 recombinase. Interestingly, despite the restriction of Rad52 mediator foci to S phase, Rad51 foci form at high levels in G1 phase. Together, these observations indicate that the recombination and checkpoint machineries promote global mobility in G1 phase, supporting the notion that recombination can occur in interphase diploids.