Cancers that are deficient in BRCA1 or BRCA2 are thought to be hypersensitive to genotoxic agents because they cannot prevent or repair DNA double strand breaks, but observations in patients suggest this dogma may no longer agree with experiment. Here, we propose that single stranded DNA underlies the hypersensitivity of BRCA deficient cancers, and that defects in double strand break repair and prevention do not. Specifically, in BRCA deficient cells, ssDNA gaps developed because replication was not effectively restrained in response to stress. In addition, we observed gaps could be suppressed by either restored fork restraint or by gap filling, both of which conferred therapy resistance in tissue culture and BRCA patient tumors. In contrast, restored double strand break repair and prevention did not confer therapy resistance when gaps were present. Critically, double strand breaks were not detected after therapy when apoptosis was inhibited, supporting a framework in which double strand breaks are not directly induced by genotoxic agents, but instead are created by cell death nucleases and are not fundamental to genotoxic agents. Together, these data indicate that ssDNA replication gaps underlie the BRCA cancer phenotype, "BRCAness," and we propose are fundamental to the mechanism-of-action of genotoxic chemotherapy.

京都大学 / 0048 / 新制・課程博士 / 博士(ｴﾈﾙｷﾞｰ科学) / 甲第20775号 / エネ博第360号 / 新制||エネ||71(附属図書館) / 京都大学大学院エネルギー科学研究科エネルギー基礎科学専攻 / (主査)教授 片平 正人, 教授 森井 孝, 教授 木下 正弘 / 学位規則第4条第1項該当 / Doctor of Energy Science / Kyoto University / DGAM

No description available.

The circular, single-stranded (ss) DNA genomes of plant viruses in the families Geminiviridae and Nanoviridae are replicated within the nucleus of a host cell by a mechanism called rolling circle replication (RCR). Although this process relies almost exclusively on the replication machinery of the host cell, initiation occurs via the interaction of the viral replication initiation protein (Rep) with regulatory DNA sequences within the viral genome. The use of a virus-based episomal amplification technology as a plant bioreactor platform exploits the process of Rep-mediated RCR for the high-level amplification of virus-based episomes in plants and subsequent expression of heterologous proteins; such an approach offers advantages over existing gene expression technologies. This PhD thesis describes research towards the development of a virus-based episomal amplification system for use in sugarcane. Such a crop is ideally suited for a plant bioreactor system due to the efficient high-level production of plant biomass and the existence of established production, harvesting and processing infrastructure. In order to rapidly assess the potential of a virus-based episomal amplification system in sugarcane, a transient assay system was established. Sugarcane callus was identified as the most suitable cell preparation; providing rapid cell regeneration, uniform experimental samples and upon isolation, total DNA suitable for Southern analysis. This assay system once established, proved effective in rapidly identifying virus-based episomes capable of undergoing RCR within sugarcane host cells. This transient assay system was then used to test the functionality of a virus-based episomal amplification system based on the ssDNA virus, Banana bunchy top virus (BBTV) in sugarcane. BBTV-based episomal amplification vectors were constructed with a reporter gene expression cassette flanked by two copies of the BBTV regulatory DNA sequences. The episomal amplification vectors were bombarded into sugarcane and banana host cells in various combinations and evidence of RCR was assessed through Southern blot analysis. RCR products were identified in banana host cells bombarded with the BBTV-based episomal amplification vectors in combination with vectors encoding BBTV Master-Rep (M Rep). RCR products were not identified within sugarcane cells bombarded with the same construct combinations. Integrated InPAct (In Plant Activation) episomal vectors based on BBTV were then employed to confirm the transient results, in addition, the functionality of an InPAct vector based on an alternate virus, Tobacco yellow dwarf virus (TYDV) was also assessed. InPAct vectors based on BBTV were constructed with an untranslatable expression cassette for integration within the sugarcane genome. Transient experiments were performed to assess the ability of BBTV M-Rep and TYDV Rep to initiate RCR of their respective InPAct vectors. Visual observation of GFP expression indicated that BBTV M-Rep was capable of initiating RCR of the BBTVbased InPAct vectors within banana host cells but no evidence was observed in sugarcane host cells. TYDV Rep was capable of initiating RCR of the TYDV-based InPAct vector within sugarcane host cells with a 100-fold increase in the number of fluorescent foci compared to cells bombarded with the TYDV InPAct vector alone. The BBTV-based InPAct vector was stably integrated within the sugarcane genome and the ability for BBTV M-Rep to initiate episome formation and RCR was assessed by Southern blot analysis. Evidence of BBTV M-Rep mediated RCR was not detected within the transgenic sugarcane bombarded with BBTV M-Rep. Transgenic sugarcane containing the TYDV-based InPAct vectors was assessed for the ability to be activated by TYDV Rep and undergo RCR. Southern blot analysis demonstrated that TYDV Rep was capable of recognising the integrated TYDVbased InPAct vector and RCR was detected within the transgenic sugarcane. The observation that episomal vectors based on TYDV were functional within sugarcane host cells and BBTV-based vectors were not, was unexpected. It had been hypothesised that an episomal vector based on a monocot-infecting virus would replicate in an alternate monocot host, while an episomal vector based on a dicot infecting virus would not. Virus replication is thought to be host-specific however most host range studies have been conducted with full length infectious clones and not deconstructed virus-based episomes. The implication that viral Reps may be functional in plant cells of non-host species was then investigated. The ability for viral Reps to recognise their cognate IR and initiate RCR of virus-based episomes in different host cells was assessed through cross-replication experiments. Four ssDNA plant viruses; BBTV, TYDV, Chloris striate mosaic virus (CSMV) and Tomato leaf curl virus - Australia (ToLCV-Au) were assessed via Southern blot analysis for their ability to initiate both autonomous replication of infectious clones and episomal amplification within three different plant hosts; tobacco, sugarcane and banana. Results from cross replication studies indicated a complex interaction between viral and host replication components. BBTV infectious clones and episomal vectors were restricted to replication within banana host cells providing a clear indication that episomal amplification vectors based on BBTV are restricted to Musa spp. BBTV M-Rep was unable to recognise the viral regulatory DNA sequences of the other three ssDNA viruses. TYDV infectious clones and episomal vectors were capable of replicating within all three host cells tested, indicating that TYDV is capable of undergoing RCR within a broad range of plant hosts. TYDV Rep was also capable of recognising the viral regulatory DNA sequences of both CSMV and BBTV given favourable conditions within specific plant hosts. Replication of the CSMV infectious clone was not detected in any of the three host cells, although fidelity of this clone requires further confirmation. CSMV episomal vectors were functional within banana host cells only, indicating that although closely related to TYDV, episomal amplification vectors based on CSMV have a restricted host range. CSMV Rep could not initiate RCR of episomal amplification vectors containing the viral regulatory DNA regions of the other three viruses in any of the plant host cells. ToLCV-Au infectious clones were capable of replicating within banana and tobacco host cells. Episomal amplification vectors based on ToLCV-Au extended the host range to sugarcane. ToLCV-Au Rep was unable to recognise the viral regulatory DNA sequences of the other three viruses in any of the plant host cells. The ability for a viral Rep to recognise its own cognate regulatory DNA sequences within alternate plant host cells is variable. Episomal amplification vectors based on TYDV and ToLCV-Au appear to be the most suitable for the further development of a virusbased bioreactor system in sugarcane. This study details the initial steps taken towards the development of a virus-based episomal amplification system in sugarcane. In doing so, fundamental knowledge into the mechanisms involved in Rep recognition of viral regulatory DNA sequences has been gathered. These research findings will provide a solid foundation for the further development of a sugarcane-based bioreactor.

Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.

Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.

Bromeliaceae é uma família importante entre as monocotiledôneas devido ao seu elevado número de espécies distribuídas no Neotrópico, e por uma riqueza de caracteres adaptativos a diferentes habitats. Flores de bromélias possuem uma grande variação morfológica, frequentemente negligenciada em estudos de morfoanatomia e de filogenia. Para uma melhor compreensão dos mecanismos de desenvolvimento que levam a essas variações, foram analisados aspectos moleculares e evolutivos do fator de transcrição MADS-box PISTILLATA (PI), a partir de sequências de transcritos isolados de inflorescências de bromélias nativas brasileiras. Sequências PI de Bromeliaceae foram comparadas com outras monocotiledôneas, com verificação de expressão em inflorescências de Tillandsia aeranthos (Loisel.) LB Sm. com o uso de hibridação in situ. Sequências de PI mostraram alta conservação, inclusive em um sítio de deleção encontrado para todos os membros analisados da família. Todos os membros da família se agruparam em um único clado em reconstruções filogenéticas. Entretanto, devido a características de taxas de mutações rápidas e divergência antiga do gene, não foi possível obter uma relação precisa entre diferentes famílias ou ordens. Todavia, PI mostrou ter potencial como ferramenta de análise filogenética para espécies proximamente relacionadas. Os transcritos de PI foram localizados principalmente em tecidos meristemáticos de regiões menos desenvolvidas de inflorescências de Tillandsia aeranthos. Flores mais desenvolvidas presentes nas inflorescências mostraram um padrão de acúmulo preferencial de transcritos PI em pétalas e sépalas, assim como esperado para flores com perianto diferenciado em sépalas e pétalas. De qualquer forma, Tillandsia aeranthos se mostrou eficiente para estudos morfoanatômicos com enfoque em desenvolvimento evolutivo. / Bromeliaceae is an important monocotyledon family due to its high number of species in the Neotropic, and a wealth of adaptive characters to different habitats. Bromeliad flowers have large morphological variations, often neglected in morpho-anatomy, floral development and phylogeny studies. For a better understanding of its floral developmental mechanisms that lead to morphological variations, molecular and evolutionary aspects of the transcription factor MADS-box PISTILLATA (PI) encoding gene isolated had been investigated in native bromeliad inflorescences. Bromeliaceae PI sequences were compared with other monocots, and the expression of these transcripts was detected in Tillandsia aeranthos (Loisel.) LB Sm. inflorescences using in situ hybridization. The PI sequences display high conservation, including a specific deletion site found in all family members. Likewise, all Bromeliaceae members grouped into a single clade in phylogeny reconstruction. However, due to rapid mutation rate and ancient divergence in PI, it was not possible to obtain a precise relationship between different monocot families and orders. Nevertheless, PI showed potential as a phylogenetic tool for analysis between closely related species. PI transcripts were located mainly in meristematic tissues from less developed regions of the inflorescences. More developed flowers showed a preferential PI transcripts accumulation in petals and stamens as expected for differentiated perianth, found in Bromeliaceae flowers. Anyway, Tillandsia aeranthos showed good potential skills for morpho-anatomy focused in evolutionary development.

No description available.

A variação do número de cópias (CNV) de genes e cromossomos é uma característica comum do genoma plástico de Leishmania major, que pode estar associada à resistência do parasita à quimioterapia das leishmanioses. Em outros eucariotos, alterações na replicação do DNA ou na resposta a danos no DNA (DDR) pode levar à CNV. Nestes organismos, o complexo de checkpoint 9-1-1 (RAD9, RAD1 e HUS1) é essencial para a detecção e a sinalização do estresse de replicação e para o recrutamento de uma apropriada DDR. Já demonstramos que L. major expressa um homólogo 9-1-1 funcional. Aqui, avaliamos a deficiência de subunidades de 9-1-1 na variação do número de cópias em células selecionadas em metotrexato (MTX), um inibidor da enzima diidrofolato redutase timidilato sintetase (DHFR-TS). A seleção em MTX facilita o isolamento de células que carregam amplificações contendo o locus da DHFR-TS. Assim, selecionamos células deficientes de HUS1 ou RAD9 para resistência ao MTX sem e com exposição previa a hidroxiureia (HU), uma droga que causa estresse de replicação por inibição da ribonucleotídeo redutase, e avaliamos o efeito da deficiência destas proteínas na CNV e no tipo de amplificação gerada. Avaliamos também o efeito da deficiência destas proteínas no processo de síntese do DNA medido pela incorporação de IdU e observamos que a deficiência destas proteínas levou a um incremento na síntese do DNA na ausência de estresse de replicação e a perfis opostos de síntese do DNA após a remoção do estresse replicativo. Análises da detecção de simples fita do DNA (ssDNA) e da histona H2A fosforilada (?H2A) como indicadores do processo de estresse de replicação e dano no DNA também foram conduzidas. Em conjunto, nossos resultados indicam que (i) os níveis alterados das proteínas HUS1 e RAD9 afetam o padrão da CNV após a seleção no MTX, assim como a natureza da amplificação; (ii) HUS1 e RAD9 parecem possuir mecanismos distintos para mediar a CNV; (iii) a função destas proteínas na CNV deve envolver o processo de replicação e (iv) HUS1 e RAD9 são requeridas para a manutenção da estabilidade genômica em Leishmania. Estes resultados contribuem para uma melhor compreensão não só da evolução da via de sinalização mediada pelo complexo de checkpoint 9-1-1 nos eucariotos, mas também da bases moleculares da plasticidade genômica e do fenômeno de amplificação gênica em Leishmania. / The copy number variation (CNV) of genes and chromosomes is a common feature of the plastic genome of Leishmania major, which is normally associated with resistance of the parasite to the chemotherapy of leishmaniasis. In other eukaryotes, alteration in DNA replication and DNA damage response (DDR) causes CNV. In these organisms, the RAD9-RAD1-HUS1 (9-1-1) checkpoint complex is essential for detection and signaling of replication stress and recruitment of an appropriate DDR. We have already demonstrated that L. major expresses a functional 9-1-1 homolog. Here we evaluated the effect of 9-1-1 subunit deficiency in CNV of cells selected in methotrexate (MTX), an inhibitor of the dihydrofolate reductase thymidylate synthetase (DHFR-TS) enzyme. Selection in MTX facilitates the isolation of cells that carry amplicons containing the DHFR-TS locus. Thus, we selected HUS1 or RAD9 deficient cells for MTX resistance without and prior exposure to hydroxyurea (HU), a drug that causes replication stress due to inhibition of ribonucleotide reductase, and evaluated not only CNV, but also the nature of the amplification generated. We also evaluated the effect of deficiency of these proteins in the DNA synthesis process measured by IdU incorporation and observed that the deficiency of these proteins led to an increase in DNA synthesis in the absence of replication stress, and to opposite profiles of DNA synthesis after removal of replicative stress. Analyzes of single-stranded DNA (ssDNA) and phosphorylated histone H2A (?H2A) as indicators of replication stress and DNA damage were also conducted in both presence and absence of replicative stress. Taken together, our results indicate that (i) altered levels of HUS1 and RAD9 proteins affect the CNV pattern after selection in MTX, as well as the nature of amplification; (ii) HUS1 and RAD9 possibly have different mechanisms to mediate CNV; (iii) the function of these proteins in CNV seems to involve replication process and (iv) HUS1 and RAD9 are required for the maintenance of genomic stability in Leishmania. These findings contribute to a better understanding not only of the evolution of the signaling pathway mediated by 9-1-1 checkpoint complex in eukaryotes, but also of the molecular basis of the genome plasticity and the gene amplification phenomenon in Leishmania.

A variação do número de cópias (CNV) de genes e cromossomos é uma característica comum do genoma plástico de Leishmania major, que pode estar associada à resistência do parasita à quimioterapia das leishmanioses. Em outros eucariotos, alterações na replicação do DNA ou na resposta a danos no DNA (DDR) pode levar à CNV. Nestes organismos, o complexo de checkpoint 9-1-1 (RAD9, RAD1 e HUS1) é essencial para a detecção e a sinalização do estresse de replicação e para o recrutamento de uma apropriada DDR. Já demonstramos que L. major expressa um homólogo 9-1-1 funcional. Aqui, avaliamos a deficiência de subunidades de 9-1-1 na variação do número de cópias em células selecionadas em metotrexato (MTX), um inibidor da enzima diidrofolato redutase timidilato sintetase (DHFR-TS). A seleção em MTX facilita o isolamento de células que carregam amplificações contendo o locus da DHFR-TS. Assim, selecionamos células deficientes de HUS1 ou RAD9 para resistência ao MTX sem e com exposição previa a hidroxiureia (HU), uma droga que causa estresse de replicação por inibição da ribonucleotídeo redutase, e avaliamos o efeito da deficiência destas proteínas na CNV e no tipo de amplificação gerada. Avaliamos também o efeito da deficiência destas proteínas no processo de síntese do DNA medido pela incorporação de IdU e observamos que a deficiência destas proteínas levou a um incremento na síntese do DNA na ausência de estresse de replicação e a perfis opostos de síntese do DNA após a remoção do estresse replicativo. Análises da detecção de simples fita do DNA (ssDNA) e da histona H2A fosforilada (?H2A) como indicadores do processo de estresse de replicação e dano no DNA também foram conduzidas. Em conjunto, nossos resultados indicam que (i) os níveis alterados das proteínas HUS1 e RAD9 afetam o padrão da CNV após a seleção no MTX, assim como a natureza da amplificação; (ii) HUS1 e RAD9 parecem possuir mecanismos distintos para mediar a CNV; (iii) a função destas proteínas na CNV deve envolver o processo de replicação e (iv) HUS1 e RAD9 são requeridas para a manutenção da estabilidade genômica em Leishmania. Estes resultados contribuem para uma melhor compreensão não só da evolução da via de sinalização mediada pelo complexo de checkpoint 9-1-1 nos eucariotos, mas também da bases moleculares da plasticidade genômica e do fenômeno de amplificação gênica em Leishmania. / The copy number variation (CNV) of genes and chromosomes is a common feature of the plastic genome of Leishmania major, which is normally associated with resistance of the parasite to the chemotherapy of leishmaniasis. In other eukaryotes, alteration in DNA replication and DNA damage response (DDR) causes CNV. In these organisms, the RAD9-RAD1-HUS1 (9-1-1) checkpoint complex is essential for detection and signaling of replication stress and recruitment of an appropriate DDR. We have already demonstrated that L. major expresses a functional 9-1-1 homolog. Here we evaluated the effect of 9-1-1 subunit deficiency in CNV of cells selected in methotrexate (MTX), an inhibitor of the dihydrofolate reductase thymidylate synthetase (DHFR-TS) enzyme. Selection in MTX facilitates the isolation of cells that carry amplicons containing the DHFR-TS locus. Thus, we selected HUS1 or RAD9 deficient cells for MTX resistance without and prior exposure to hydroxyurea (HU), a drug that causes replication stress due to inhibition of ribonucleotide reductase, and evaluated not only CNV, but also the nature of the amplification generated. We also evaluated the effect of deficiency of these proteins in the DNA synthesis process measured by IdU incorporation and observed that the deficiency of these proteins led to an increase in DNA synthesis in the absence of replication stress, and to opposite profiles of DNA synthesis after removal of replicative stress. Analyzes of single-stranded DNA (ssDNA) and phosphorylated histone H2A (?H2A) as indicators of replication stress and DNA damage were also conducted in both presence and absence of replicative stress. Taken together, our results indicate that (i) altered levels of HUS1 and RAD9 proteins affect the CNV pattern after selection in MTX, as well as the nature of amplification; (ii) HUS1 and RAD9 possibly have different mechanisms to mediate CNV; (iii) the function of these proteins in CNV seems to involve replication process and (iv) HUS1 and RAD9 are required for the maintenance of genomic stability in Leishmania. These findings contribute to a better understanding not only of the evolution of the signaling pathway mediated by 9-1-1 checkpoint complex in eukaryotes, but also of the molecular basis of the genome plasticity and the gene amplification phenomenon in Leishmania.