Functional compartmentalization of Rad9 and Hus1 reveals diverse assembly of the 9-1-1 complex components during the DNA damage response in Leishmania

The Rad9-Rad1-Hus1 (9-1-1) complex is a key component in the coordination of DNA damage sensing, cell cycle progression and DNA repair pathways in eukaryotic cells. This PCNA-related trimer is loaded onto RPA-coated single stranded DNA and interacts with ATR kinase to mediate effective checkpoint signaling to halt the cell cycle and to promote DNA repair. Beyond these core activities, mounting evidence suggests that a broader range of functions can be provided by 9-1-1 structural diversification. The protozoan parasite Leishmania is an early-branching eukaryote with a remarkably plastic genome, which hints at peculiar genome maintenance mechanisms. Here, we investigated the existence of homologs of the 9-1-1 complex subunits in L. major and found that LmRad9 and LmRad1 associate with chromatin in response to replication stress and form a complex in vivo with LmHus1. Similar to LmHus1, LmRad9 participates in telomere homeostasis and in the response to both replication stress and double strand breaks. However, LmRad9 and LmHus1-deficient cells present markedly opposite phenotypes, which suggest their functional compartmentalization. We show that some of the cellular pool of LmRad9 forms an alternative complex and that some of LmHus1 exists as a monomer. We propose that the diverse assembly of the Leishmania 9-1-1 subunits mediates functional compartmentalization, which has a direct impact on the response to genotoxic stress

Genome duplication in Leishmania major relies on persistent subtelomeric DNA replication

DNA replication is needed to duplicate a cell’s genome in S phase and segregate it during cell division. Previous work in Leishmania detected DNA replication initiation at just a single region in each chromosome, an organisation predicted to be insufficient for complete genome duplication within S phase. Here, we show that acetylated histone H3 (AcH3), base J and a kinetochore factor co-localise in each chromosome at only a single locus, which corresponds with previously mapped DNA replication initiation regions and is demarcated by localised G/T skew and G4 patterns. In addition, we describe previously undetected subtelomeric DNA replication in G2/M and G1-phase-enriched cells. Finally, we show that subtelomeric DNA replication, unlike chromosome-internal DNA replication, is sensitive to hydroxyurea and dependent on 9-1-1 activity. These findings indicate that Leishmania’s genome duplication programme employs subtelomeric DNA replication initiation, possibly extending beyond S phase, to support predominantly chromosome-internal DNA replication initiation within S phase

Conditional genome engineering reveals canonical and divergent roles for the Hus1 component of the 9-1-1 complex in the maintenance of the plastic genome of Leishmania.

Leishmania species are protozoan parasites whose remarkably plastic genome limits the establishment of effective genetic manipulation and leishmaniasis treatment. The strategies used by Leishmania to maintain its genome while allowing variability are not fully understood. Here, we used DiCre-mediated conditional gene deletion to show that HUS1, a component of the 9-1-1 (RAD9- RAD1-HUS1) complex, is essential and is required for a G2/M checkpoint. By analyzing genome wide instability in HUS1 ablated cells, HUS1 is shown to have a conserved role, by which it preserves genome stability, and also a divergent role, by which it promotes genome variability. These roles of HUS1 are related to distinct patterns of formation and resolution of single-stranded DNA and γH2A, throughout the cell cycle. Our findings suggest that Leishmania 9-1-1 subunits have evolved to co-opt canonical genomic maintenance and genomic variation functions. Hence, this study reveals a pivotal function of HUS1 in balancing genome stability and transmission in Leishmania. These findings may be relevant to understanding the evolution of genome maintenance and plasticity in other pathogens and eukaryote

Deficiency of checkpoint proteins HUS1 and RAD9 promotes copy number variation in the Leishmania major genome

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

Deficiency of checkpoint proteins HUS1 and RAD9 promotes copy number variation in the Leishmania major genome

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