Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection

DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11–Rad50–Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5'-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3' overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination

Stepwise 5' DNA end-specific resection of DNA breaks by the Mre11-Rad50-Xrs2 and Sae2 nuclease ensemble

To repair DNA double-strand breaks by homologous recombination, the 5′-terminated DNA strands must first be resected to produce 3′ overhangs. Mre11 from Saccharomyces cerevisiae is a 3′ → 5′ exonuclease that is responsible for 5′ end degradation in vivo. Using plasmid-length DNA substrates and purified recombinant proteins, we show that the combined exonuclease and endonuclease activities of recombinant MRX-Sae2 preferentially degrade the 5′-terminated DNA strand, which extends beyond the vicinity of the DNA end. Mechanistically, Rad50 restricts the Mre11 exonuclease in an ATP binding-dependent manner, preventing 3′ end degradation. Phosphorylated Sae2, along with stimulating the MRX endonuclease as shown previously, also overcomes this inhibition to promote the 3′ → 5′ exonuclease of MRX, which requires ATP hydrolysis by Rad50. Our results support a model in which MRX-Sae2 catalyzes 5′-DNA end degradation by stepwise endonucleolytic DNA incisions, followed by exonucleolytic 3′ → 5′ degradation of the individual DNA fragments. This model explains how both exonuclease and endonuclease activities of Mre11 functionally integrate within the MRX-Sae2 ensemble to resect 5′-terminated DNA

Qualidade microbiológica de fórmulas infantis administradas em hospital público do município de Campinas, São Paulo

A qualidade microbiológica em lactários é um item que merece toda a atenção e deve ser mantida de forma rigorosa. Considerando a população alvo desses serviços e o ambiente em que estão inseridos, sabe-se que são locais de alto risco para contaminações microbiológicas e as consequências destas podem ser fatais. Assim, o objetivo deste estudo foi verificar a qualidade microbiológica de fórmulas infantis utilizadas para alimentação de crianças internadas na unidade de pediatria de um hospital público do município de Campinas – SP. Foram realizadas análises microbiológicas de bactérias aeróbias mesófilas totais, coliformes totais e termotolerantes e Estafilococos coagulase positiva das fórmulas infantis, comparando-se resultados com os valores especificados na Resolução da Diretoria Colegiada no 12, de 02 de janeiro de 2001 (RDC 12/2001), da Agência de Vigilância Sanitária. Das 26 amostras de fórmulas infantis, 88,5% atenderam aos parâmetros para oferecimento para menores de 1 ano de vida, e 92,3% atenderam para as exigências para pacientes com mais de 1 ano de vida, segundo a RDC 12/2001. Porém, quando observados paralelamente adequação a parâmetros desta legislação e a recomendação da Food and Drug Administration (2006) para microrganismos aeróbios mesófilos, apenas 46,2% apresentavam contagens dos microrganismos avaliados dentro dos limites tolerados

Qualidade Microbiológica De Fórmulas Infantis Administradas Em Hospital Público Do Município De Campinas, São Paulo

A qualidade microbiológica em lactários é um item que merece toda a atenção e deve ser mantida de forma rigorosa. Considerando a população alvo desses serviços e o ambiente em que estão inseridos, sabe-se que são locais de alto risco para contaminações microbiológicas e as consequências destas podem ser fatais. Assim, o objetivo deste estudo foi verificar a qualidade microbiológica de fórmulas infantis utilizadas para alimentação de crianças internadas na unidade de pediatria de um hospital público do município de Campinas – SP. Foram realizadas análises microbiológicas de bactérias aeróbias mesófilas totais, coliformes totais e termotolerantes e Estafilococos coagulase positiva das fórmulas infantis, comparando-se resultados com os valores especificados na Resolução da Diretoria Colegiada no 12, de 02 de janeiro de 2001 (RDC 12/2001), da Agência de Vigilância Sanitária. Das 26 amostras de fórmulas infantis, 88,5% atenderam aos parâmetros para oferecimento para menores de 1 ano de vida, e 92,3% atenderam para as exigências para pacientes com mais de 1 ano de vida, segundo a RDC 12/2001. Porém, quando observados paralelamente adequação a parâmetros desta legislação e a recomendação da Food and Drug Administration (2006) para microrganismos aeróbios mesófilos, apenas 46,2% apresentavam contagens dos microrganismos avaliados dentro dos limites tolerados.211387-394Segurança Alimentar

The MRE11 complex: a versatile toolkit for the repair of broken DNA

When DNA breaks, the ends need to be stabilized and processed to facilitate subsequent repair, which can occur by either direct but error-prone end-joining with another broken DNA molecule or a more accurate homology-directed repair by the recombination machinery. At the same time, the presence of broken DNA triggers a signaling cascade that regulates the repair events and cellular progression through the cell cycle. The MRE11 nuclease, together with RAD50 and NBS1 forms a complex termed MRN that participates in all these processes. Although MRE11 was first identified more than 20 years ago, deep insights into its mechanism of action and regulation are much more recent. Here we review how MRE11 functions within MRN, and how the complex is further regulated by CtIP and its phosphorylation in a cell cycle dependent manner. We describe how RAD50, NBS1 and CtIP convert MRE11, exhibiting per se a 3′→5′ exonuclease activity, into an ensemble that instead degrades primarily the 5′-terminated strand by endonucleolytic cleavage at DNA break sites to generate 3′ overhangs, as required for the initiation of homologous recombination. The unique mechanism of DNA end resection by MRN-CtIP makes it a very flexible toolkit to process DNA breaks with a variety of secondary structures and protein blocks. Such a block can also be the Ku heterodimer, and emerging evidence suggests that MRN-CtIP may often need to remove Ku from DNA ends before initiating homologous recombination. Misregulation of DNA break repair results in mutations and chromosome rearrangements that can drive cancer development. Therefore, a detailed understanding of the underlying processes is highly relevant for human health

Physiological protein blocks direct the Mre11-Rad50-Xrs2 and Sae2 nuclease complex to initiate DNA end resection

DNA double-strand break repair by homologous recombination is initiated by DNA end resection, which is commenced by the Mre11–Rad50–Xrs2 complex and Sae2 in yeast. Here we report that the nonhomologous end joining factor Ku limits the exonuclease activity of Mre11 and promotes its endonuclease to cleave 5′-terminated DNA strands at break sites. Following initial endonucleolytic cleavage past the obstacle, Exo1 specifically extends the resection track, leading to the generation of long 3′ overhangs that are required for homologous recombination. These experiments provide mechanistic insights into how short-range and long-range DNA end resection enzymes overcome obstacles near broken DNA ends to initiate recombination.ISSN:0890-9369ISSN:1549-547

Crossover or non-crossover outcomes: tailored processing of homologous recombination intermediates

DNA breaks may arise accidentally in vegetative cells or in a programmed manner in meiosis. The usage of a DNA template makes homologous recombination potentially error-free, however, recombination is not always accurate. Cells possess a remarkable capacity to tailor processing of recombination intermediates to fulfill a particular need. Vegetatively growing cells aim to maintain genome stability and therefore repair accidental breaks largely accurately, using sister chromatids as templates, into mostly non-crossovers products. Recombination in meiotic cells is instead more likely to employ homologous chromosomes as templates and result in crossovers to allow proper chromosome segregation and promote genetic diversity. Here we review models explaining the processing of recombination intermediates in vegetative and meiotic cells and its regulation, with a focus on MLH1-MLH3-dependent crossing-over during meiotic recombination

Single molecule experiments highlight the role of NBS1 and Xrs2 in DNA tethering by MRN and MRX

(Poster presentation at 14th EBSA congress, July 31 – August 4, 2023, Stockholm, Sweden