53BP1 Enforces Distinct Pre- and Post-resection Blocks on Homologous Recombination.

53BP1 activity drives genome instability and lethality in BRCA1-deficient mice by inhibiting homologous recombination (HR). The anti-recombinogenic functions of 53BP1 require phosphorylation-dependent interactions with PTIP and RIF1/shieldin effector complexes. While RIF1/shieldin blocks 5'-3' nucleolytic processing of DNA ends, it remains unclear how PTIP antagonizes HR. Here, we show that mutation of the PTIP interaction site in 53BP1 (S25A) allows sufficient DNA2-dependent end resection to rescue the lethality of BRCA1Δ11 mice, despite increasing RIF1 "end-blocking" at DNA damage sites. However, double-mutant cells fail to complete HR, as excessive shieldin activity also inhibits RNF168-mediated loading of PALB2/RAD51. As a result, BRCA1Δ1153BP1S25A mice exhibit hallmark features of HR insufficiency, including premature aging and hypersensitivity to PARPi. Disruption of shieldin or forced targeting of PALB2 to ssDNA in BRCA1D1153BP1S25A cells restores RNF168 recruitment, RAD51 nucleofilament formation, and PARPi resistance. Our study therefore reveals a critical function of shieldin post-resection that limits the loading of RAD51.We thank Anthony Tubbs for comments on the paper; Jennifer Mehalko and Dom Esposito (Protein Expression Laboratory, Frederick National Laboratory for Cancer Research) for transgenic constructs; Karim Baktiar, Diana Haines, and Elijah Edmonson (Pathology/Histotechnology Laboratory, Frederick National Laboratory for Cancer Research) for rodent necropsy, pathology analysis, and imaging; Joseph Kalen and Nimit Patel (Small Animal Imaging Program, Frederick National Laboratory for Cancer Research) for X-ray computed tomography (CT) scan imaging; Jennifer Wise and Kelly Smith for assistance with animal work; Davide Robbiani and Kai Ge for antibodies; Dan Durocher for shieldin constructs; David Goldstein and the CCR Genomics core for sequencing support; and Neil Johnson for discussions. Research in the J.M.S. laboratory is supported by NIH grant R01CA197506. Research in the N.M. laboratory is supported by NIH grant R01 227001. The A.N. laboratory is supported by the Intramural Research Program of the NIH, an Ellison Medical Foundation Senior Scholar in Aging Award (AG-SS-2633-11), the Department of Defense Idea Expansion (W81XWH-15-2-006) and Breakthrough (W81XWH-16-1-599) Awards, the Alex's Lemonade Stand Foundation Award, and an NIH Intramural FLEX Award.S

Nuclear DNA Replication in Trypanosomatids:There Are No Easy Methods for Solving Difficult Problems

In trypanosomatids, etiological agents of devastating diseases, replication is robust and finely controlled to maintain genome stability and function in stressful environments. However, these parasites encode several replication protein components and complexes that show potentially variant composition compared with model eukaryotes. This review focuses on the advances made in recent years regarding the differences and peculiarities of the replication machinery in trypanosomatids, including how such divergence might affect DNA replication dynamics and the replication stress response. Comparing the DNA replication machinery and processes of parasites and their hosts may provide a foundation for the identification of targets that can be used in the development of chemotherapies to assist in the eradication of diseases caused by these pathogens

EXO1 protects BRCA1-deficient cells against toxic DNA lesions

Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors

Characterization of RPA-1-telomere interaction in Trypanosoma cruzi.

O complexo telomérico, responsável pela integridade genômica, é formado pela interação de DNA com proteínas, que são responsáveis pela proteção desses terminais. O complexo RPA de eucariotos compreende um heterotrímero, que cumpre diversas funções vitais na célula, sendo uma peça fundamental na replicação, reparo e recombinação. A ausência de homólogos de proteínas que protegem o telômero em T. cruzi nos fez investigar se o complexo RPA poderia cumprir essa função. Assim, este trabalho teve como objetivo caracterizar a interação TcRPA-1-telômero. Conseguimos verificar a interação in vitro e in vivo da RPA-1 com o telômero em epimastigotas e tripomastigotas. A ausência de homólogos de proteínas que interagem com o overhang telomérico em tripanosomas, a interação específica RPA-1-telômero, e a sua presença nos telômeros da forma de vida não replicativa, bem como as peculiaridades estruturais da TcRPA-1, levam-nos a propor que essa proteína pode estar envolvida com a proteção dos telomérica neste organismo.The telomeric complex, responsible for genomic integrity and stability , is formed by the interaction of DNA with proteins that are responsible for maintaining and protecting these terminals. The eukaryotic RPA complex comprises a heterotrimer, which fulfills several vital functions in the cell , being a fundamental player in replication , repair and recombination. The absence of homologous proteins that protect telomeres in Trypanosoma cruzi lead us to hypothesize that RPA complex could fulfill this function. Thus, this study aimed to characterize TcRPA-1-telomere interaction. We could verify in vitro and in vivo interaction of RPA - 1 with telomeres in epimastigote and trypomastigote lifeforms. The absence of homologs of proteins that interact with telomeric overhang in trypanosomes, the specific interaction RPA-1- telomere , its presence in non- replicative lifeform as well as structural peculiarities of TcRPA-1, lead us to propose that this protein may be involved in telomere protection in this organism

Functional analysis of RPA complex in trypanosomatids and its involvement with telomeric DNA.

Trypanosoma cruzi e Trypanosoma brucei são protozoários parasitos responsáveis pela doença de Chagas e pela doença do sono, as quais resultam em um elevado número de mortes anualmente. Esses parasitos possuem um ciclo de vida complexo que alterna entre formas de vida replicativas e não replicativas. Apesar da caracterização de um grande número de vias moleculares nestes organismos, ainda existem muitas lacunas na compreensão dos processos que coordenam o metabolismo do DNA. A proteína Replication Protein A (RPA), principal ligante de fita simples de DNA de eucariotos, é um complexo heterotrimérico formado por três subunidades RPA-1, RPA2 e RPA-3 que participa em vários processos fundamentais como replicação, reparo e sinalização de checkpoint. A RPA de tripanossomatídeos apresenta peculiaridades estruturais significativas em comparação a outros eucariotos, como a falta de domínio de DBD-F (70N) que interage com proteínas majoritariamente envolvidas na resposta a danos no DNA (DDR) e substituições em resíduos de aminoácidos em regiões conservadas, levantando questões sobre a conservação de funções canônicas descritas em mamíferos e leveduras. Neste trabalho, mostramos que o RPA de tripanossomatídeos pode interagir com DNA fita simples e é de fato importante para replicação e resposta a danos de DNA. Além disso, conseguimos encontrar diversas novas características nas RPA de tripanossomatídeos, como a descoberta de (i) modificações pós-traducionais não descritas (ii) uma nova proteína RPA-like que parece ser exclusiva de tripanossomatídeos interagindo com o complexo RPA (iii) um processo de exportação nuclear ciclo de vida dependente e (iv) alta afinidade pelo DNA de fita simples telomérico rico em G.Trypanosoma cruzi and Trypanosoma brucei are parasitic protozoa responsible for causing Chagas disease and sleeping sickness that result in a high number of deaths annually. These parasites present a complex life cycle that alternates between replicative and non-replicative lifeforms. Despite the characterization of a great number of molecular pathways, there are still many gaps in the understanding of the processes that coordinate the DNA metabolism of these organisms. Replication protein A (RPA), the major eukaryotic single-stranded binding protein, is a well-known heterotrimeric complex formed by three subunits RPA-1, RPA2, and RPA-3 that participates in various vital functions during replication, repair, and checkpoint signaling. RPA from trypanosomatids presents significant structural peculiarities compared to other eukaryotes such as the lacking of DBD-F (70N) domain that interacts with proteins majorly involved in DNA damage response (DDR) pathways and amino acids substitutions in conserved regions, raising questions regarding the conservation of canonical functions described in mammals and yeast. In this work, we show that RPA from trypanosomatids can interact with single-stranded DNA and is indeed important for replication and DNA damage response pathways. Moreover, we could find new features concerning trypanosomatids RPA such as the discovery of (i) non-described post-translation modifications (ii) a new RPA-like protein that seems to be exclusive of trypanosomatids interacting with RPA complex (iii) a nucleus-cytoplasm shuttle that is lifecycle dependent and (iv) high affinity for G-rich telomeric single stranded DNA

Clonagem, expressão e purificação de três mutantes truncados da proteína LaRPA-1 de Leishmania amazonensis

A leishmaniose é uma doença que atinge milhões de pessoas no mundo e, de acordo com a FUNASA, está se espalhando por todas as regiões do Brasil. O tratamento desta zoonose é ineficaz, pois os fármacos utilizados apresentam muitos efeitos colaterais e colaboram com o aparecimento de parasitas e vetores resistentes. Portanto um maior conhecimento sobre a biologia molecular destes protozoários poderá facilitar o descobrimento de novas terapias e desenvolvimento de drogas antiparasitárias. O complexo telomérico é formado pela interação de DNA com proteínas, sendo que estas últimas podem também interagir entre si. A proteína RPA de eucariotos compreende um complexo trimérico subdividido em três subunidades. Este cumpre independentemente ou juntamente com outras proteínas diversas funções vitais para a célula, entre elas, auxiliar na manutenção dos telômeros e ajudar a recrutar a telomerase. Além disso, ela parece ser um dos principais componentes do complexo de proteínas que interagem com o terminal simples fita telomérico de protozoários tripanosomatídeos. Curiosamente, em Leishmania spp., as subunidades 2 e 3, ao contrário da subunidade 1 da RPA, não fazem parte do complexo telomérico. Portanto a LaRPA-1 pode apresentar comportamentos peculiares quando comparada a RPA dos outros eucariotos, e se tornar um importante alvo ao se estudar a biologia molecular do parasita. Este trabalho tem como objetivo clonar, expressar e purificar os três diferentes mutantes truncados da proteína LaRPA-1 para, futuramente, utilizar-se estas proteínas recombinantes como ligantes em ensaios de interação proteína:proteína, visando mapear possíveis sítios ou domínios na proteína LaRPA-1.O gene que codifica LaRPA-1 já encontrava-se clonado e sequenciado no laboratório de Telômeros da UNESP de Botucatu....(Resumo completo, clicar acesso eletrônico abaixo

Metagenomic assembly and draft genome sequence of an Uncharacterized prevotella sp. from Nelore rumen

Prevotella is one of the most abundant genera in bovine rumen, although no genome has yet been assembled by a metagenomics approach applied to Brazilian Nelore. We report the draft genome sequence of Prevotella sp., comprising 2,971,040 bp, obtained using the Illumina sequencing platform. This genome includes 127 contigs and presents a low 48% GC

Glyceraldehyde 3-phosphate dehydrogenase-telomere association correlates with redox status in trypanosoma cruzi

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is a classical metabolic enzyme involved in energy production and plays a role in additional nuclear functions, including transcriptional control, recognition of misincorporated nucleotides in DNA and maintenance of telomere structure. Here, we show that the recombinant protein T. cruzi GAPDH (rTcGAPDH) binds single-stranded telomeric DNA. We demonstrate that the binding of GAPDH to telomeric DNA correlates with the balance between oxidized and reduced forms of nicotinamide adenine dinucleotides (NAD+/NADH). We observed that GAPDH-telomere association and NAD+/NADH balance changed throughout the T. cruzi life cycle. For example, in replicative epimastigote forms of T. cruzi, which show similar intracellular concentrations of NAD+ and NADH, GAPDH binds to telomeric DNA in vivo and this binding activity is inhibited by exogenous NAD+. In contrast, in the T. cruzi non-proliferative trypomastigote forms, which show higher NAD+ concentration, GAPDH was absent from telomeres. In addition, NAD+ abolishes physical interaction between recombinant GAPDH and synthetic telomere oligonucleotide in a cell free system, mimicking exogenous NAD+ that reduces GAPDH-telomere interaction in vivo. We propose that the balance in the NAD+/NADH ratio during T. cruzi life cycle homeostatically regulates GAPDH telomere association, suggesting that in trypanosomes redox status locally modulates GAPDH association with telomeric DNA.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP

Estilos de vida de pacientes hipertensos atendidos com a Estratégia de Saúde Familiar

Objective. To describe hypertensive patients attended with the Family Health strategy lifestyle. Methodology. Cross sectional study, carried out between October 2009 and January 2010. 273 hypertensive patients randomly chosen from the Family Health program attended by three areas of Piraí/RJ (Brazil) participated. For data collection, the fantastic lifestyle questionnaire was used. Results. From the studied sample: 61% were women, 56% were 60 and over years of age, 81% had a low level of education and 74% reported having low family income. The most frequent risk factors were: overweight or obesity (72%) and diabetes mellitus (37%). Lifestyle scores were: 13% excellent, 55% very good, 27% good, and 4% poor. Conclusion. Even though lifestyle was considered satisfactory, conditions and health profiles indicate that there are still cardiovascular risk factors.Objetivo. Descrever os estilos de vida dos pacientes hipertensos atendidos com a estratégia de Saúde Familiar. Metodologia. De outubro de 2009 a janeiro de 2010, levou-se a cabo um estudo transversal, no que participaram 273 pacientes hipertensos selecionados aleatoriamente do programa de Saúde Familiar que atende três áreas de Piraí/RJ (Brasil). Para a colheita de dados se utilizou o questionário Estilo de Vida Fantástico. Resultados. Da mostra de estudo: o 61% eram mulheres, o 56% tinha 60 e mais anos, o 81% possuía sob nível de educação e 74% reportou ter baixos rendimentos familiares. Os fatores de risco mais frequentes foram o sobrepeso ou obesidade (72%) e a diabete mellitus (37%). A qualificação do estilo de vida foi: 13% excelente, muito bom 55%, bom 27% e regular 4%. Conclusão. Ainda que o estilo de vida se considerou satisfatório, as condições e o perfil de saúde indicam a persistência de fatores de risco de doença cardiovascular.Objetivo. Describir los estilos de vida de los pacientes hipertensos atendidos con la Estrategia de Salud de La Familia. Metodología. De octubre de 2009 a enero de 2010, se llevó a cabo un estudio transversal, en el que participaron 273 pacientes hipertensos seleccionados aleatoriamente del programa de salud familiar que atiende tres áreas de Piraí/RJ. Para la recolección de datos se utilizó el cuestionario Estilo de Vida Fantástico. Resultados. De la muestra de estudio: el 61% eran mujeres, el 56% con 60 y más años, el 81% tenía bajo nivel de educación y 74% con bajos ingresos familiares. Los factores de riesgo más frecuentes fueron El sobrepeso u obesidad (72%) y la diabetes mellitus (37%). La calificación del estilo de vida fue: 13% excelente, 55% muy bueno, 27% bueno y regular 4%. Conclusión. Aunque el estilo de invida se consideró satisfactorio, las condiciones socioeconmicas y el perfil de salud indican la persistencia de factores de riesgo de enfermedad cardiovascular