ATR and H2AX Cooperate in Maintaining Genome Stability under Replication Stress

Chromosomal abnormalities are frequently caused by problems encountered during DNA replication. Although the ATR-Chk1 pathway has previously been implicated in preventing the collapse of stalled replication forks into double-strand breaks (DSB), the importance of the response to fork collapse in ATR-deficient cells has not been well characterized. Herein, we demonstrate that, upon stalled replication, ATR deficiency leads to the phosphorylation of H2AX by ATM and DNA-PKcs and to the focal accumulation of Rad51, a marker of homologous recombination and fork restart. Because H2AX has been shown to play a facilitative role in homologous recombination, we hypothesized that H2AX participates in Rad51-mediated suppression of DSBs generated in the absence of ATR. Consistent with this model, increased Rad51 focal accumulation in ATR-deficient cells is largely dependent on H2AX, and dual deficiencies in ATR and H2AX lead to synergistic increases in chromatid breaks and translocations. Importantly, the ATM and DNA-PK phosphorylation site on H2AX (Ser¹³⁹) is required for genome stabilization in the absence of ATR; therefore, phosphorylation of H2AX by ATM and DNA-PKcs plays a pivotal role in suppressing DSBs during DNA synthesis in instances of ATR pathway failure. These results imply that ATR-dependent fork stabilization and H2AX/ATM/DNA-PKcs-dependent restart pathways cooperatively suppress double-strand breaks as a layered response network when replication stalls.Genome maintenance prevents mutations that lead to cancer and age-related diseases. A major challenge in preserving genome integrity occurs in the simple act of DNA replication, in which failures at numerous levels can occur. Besides the mis-incorporation of nucleotides, it is during this phase of the cell cycle that the relatively stable double-stranded nature of DNA is temporarily suspended at the replication fork, a structure that is susceptible to collapse into DSBs.² Replication fork stability is maintained by a variety of mechanisms, including activation of the ATR-dependent checkpoint pathway.The ATR pathway is activated upon the generation and recognition of extended stretches of single-stranded DNA at stalled replication forks (1-4). Genome maintenance functions for ATR and orthologs in yeast were first indicated by increased chromatid breaks in ATR^-/- cultured cells (5) and by the “cut” phenotype observed in Mec1 (Saccharomyces cerevisiae) and Rad3 (Schizosaccharomyces pombe) mutants (6-9). Importantly, subsequent studies in S. cerevisiae demonstrated that mutation of Mec1 or the downstream checkpoint kinase Rad53 led to increased chromosome breaks at regions of the genome that are inherently difficult to replicate (10), and a decreased ability to reinitiate replication fork progression following DNA damage or deoxyribonucleotide depletion (11-14).In vertebrates, similar replication fork stabilizing functions have been demonstrated for ATR and the downstream protein kinase Chk1 (15-20). Several possible mechanisms have been put forward to explain how ATR-Chk1 and orthologous pathways in yeast maintain replication fork stability, including maintenance of replicative polymerases (α, δ, and ε) at forks (17, 21), regulation of branch migrating helicases, such as Blm (22-25), and regulation of homologous recombination, either positively or negatively (26-29).Consistent with the role of the ATR-dependent checkpoint in replication fork stability, common fragile sites, located in late-replicating regions of the genome, are significantly more unstable (5-10-fold) in the absence of ATR or Chk1 (19, 20). Because these sites are favored regions of instability in oncogene-transformed cells and preneoplastic lesions (30, 31), it is possible that the increased tumor incidence observed in ATR haploinsufficient mice (5, 32) may be related to subtle increases in genomic instability. Together, these studies indicate that maintenance of replication fork stability may contribute to tumor suppression.It is important to note that prevention of fork collapse represents an early response to problems occurring during DNA replication. In the event of fork collapse into DSBs, homologous recombination (HR) has also been demonstrated to play a key role in genome stability during S phase by catalyzing recombination between sister chromatids as a means to re-establish replication forks (33). Importantly, a facilitator of homologous recombination, H2AX, has been shown to be phosphorylated under conditions that cause replication fork collapse (18, 34).Phosphorylation of H2AX occurs predominantly upon DSB formation (34-38) and has been reported to require ATM, DNA-PKcs, or ATR, depending on the context (37-42). Although H2AX is not essential for HR, studies have demonstrated that H2AX mutation leads to deficiencies in HR (43, 44), and suppresses events associated with homologous recombination, such as the focal accumulation of Rad51, BRCA1, BRCA2, ubiquitinated-FANCD2, and Ubc13-mediated chromatin ubiquitination (43, 45-51). Therefore, through its contribution to HR, it is possible that H2AX plays an important role in replication fork stability as part of a salvage pathway to reinitiate replication following collapse.If ATR prevents the collapse of stalled replication forks into DSBs, and H2AX facilitates HR-mediated restart, the combined deficiency in ATR and H2AX would be expected to dramatically enhance the accumulation of DSBs upon replication fork stalling. Herein, we utilize both partial and complete elimination of ATR and H2AX to demonstrate that these genes work cooperatively in non-redundant pathways to suppress DSBs during S phase. As discussed, these studies imply that the various components of replication fork protection and regeneration cooperate to maintain replication fork stability. Given the large number of genes involved in each of these processes, it is possible that combined deficiencies in these pathways may be relatively frequent in humans and may synergistically influence the onset of age-related diseases and cancer.     

Intracellular fate of Francisella tularensis within arthropod-derived cells

Since transmission of Francisella tularensis into the mammalian host occurs via arthropod vectors such as ticks, mosquitoes, horseflies and deerflies, recent studies have established Drosophila melanogaster as an arthropod vector model system. Nothing is known about the intracellular fate of F. tularensis within arthropod‐derived cells, and the role of this host‐parasite adaptation in the evolution of this pathogen to infect mammals. In this report, we explored intracellular trafficking of F. tularensis ssp. novicida in D. melanogaster‐derived S2 cells. First, we show that similar to the F. tularensis ssp. holarctica‐derived LVS strain, F. tularensis ssp. novicida is highly infectious, replicates exponentially within S2 cells and within adult flies, and is fatal to adult fruit flies in a dose‐dependent manner, while the iglC, iglD and mglA mutants are defective. Using electron and fluorescence microscopy‐based phagosome integrity assays, we show that the wild‐type strain escapes into the cytosol of S2 cells within 30–60 min post infection and by 6 h, 90% were cytosolic. In contrast, approximately 40–50% of the iglC and iglD mutants escape into the cytosol by 6 h while the other subpopulation becomes enclosed within multilamellar vesicles (MLVs). Pre‐treatment of S2 cells with the autophagy inhibitor methyl adenine blocks formation of the MLVs and all the vacuolar subpopulation of the iglC and iglD mutant bacteria become enclosed within single membrane‐surrounded vacuoles. Endocytic trafficking studies of F. tularensis within S2 cells show transient colocalization of the bacterial phagosome with D. melanogaster LAMP2–GFP fusion but not with lysosomes pre‐loaded with fluorescent dextran. Our data show that MLVs harbouring the iglC mutant acquire Lamp2 and dextran while MLVs harbouring the iglD mutant exclude these late endosomal and lysosomal markers. Our data indicate crucial differences in the role of the pathogenicity island‐encoded proteins in modulating intracellular trafficking within human macrophages and arthropod vector‐derived cells.     

Molecular bases of proliferation of Francisella tularensis in arthropod vectors

Arthropod vectors are important vehicles for transmission of Francisella tularensis between mammals, but very little is known about the F. tularensis–arthropod vector interaction. Drosophila melanogaster has been recently developed as an arthropod vector model for F. tularensis. We have shown that intracellular trafficking of F. tularensis within human monocytes‐derived macrophages and D. melanogaster‐derived S2 cells is very similar. Within both evolutionarily distant host cells, the Francisella‐containing phagosome matures to a late endosome‐like phagosome with limited fusion to lysosomes followed by rapid bacterial escape into the cytosol where the bacterial proliferate. To decipher the molecular bases of intracellular proliferation of F. tularensis within arthropod‐derived cells, we screened a comprehensive library of mutants of F. tularensis ssp. novicida for their defect in intracellular proliferation within D. melanogaster‐derived S2 cells. Our data show that 394 genes, representing 22% of the genome, are required for intracellular proliferation within D. melanogaster‐derived S2 cells, including many of the Francisella Pathogenicity Island (FPI) genes that are also required for proliferation within mammalian macrophages. Functional gene classes that exhibit growth defect include metabolic (25%), FPI (2%), type IV pili (1%), transport (16%) and DNA modification (5%). Among 168 most defective mutants in intracellular proliferation in S2 cells, 80 are defective in lethality and proliferation within adult D. melanogaster. The observation that only 135 of the 394 mutants that are defective in S2 cells are also defective in human macrophages indicates that F. tularensis utilize common as well as distinct mechanisms to proliferate within mammalian and arthropod cells. Our studies will facilitate deciphering the molecular aspects of F. tularensis–arthropod vector interaction and its patho‐adaptation to infect mammals.     

Proteomic analysis of microparticles isolated from malaria positive blood samples

Background

Malaria continues to be a great public health concern due to the significant mortality and morbidity associated with the disease especially in developing countries. Microparticles (MPs), also called plasma membrane derived extracellular vesicles (PMEVs) are subcellular structures that are generated when they bud off the plasma membrane. They can be found in healthy individuals but the numbers tend to increase in pathological conditions including malaria. Although, various studies have been carried out on the protein content of specific cellular derived MPs, there seems to be paucity of information on the protein content of circulating MPs in malaria and their association with the various signs and symptoms of the disease. The aim of this study was therefore to carry out proteomic analyses of MPs isolated from malaria positive samples and compare them with proteins of MPs from malaria parasite culture supernatant and healthy controls in order to ascertain the role of MPs in malaria infection.

Methods

Plasma samples were obtained from forty-three (43) malaria diagnosed patients (cases) and ten (10) healthy individuals (controls). Malaria parasite culture supernatant was obtained from our laboratory and MPs were isolated from them and confirmed using flow cytometry. 2D LC-MS was done to obtain their protein content. Resultant data were analyzed using SPSS Ver. 21.0 statistical software, Kruskal Wallis test and Spearman’s correlation coefficient r.

Results

In all, 1806 proteins were isolated from the samples. The MPs from malaria positive samples recorded 1729 proteins, those from culture supernatant were 333 while the control samples recorded 234 proteins. The mean number of proteins in MPs of malaria positive samples was significantly higher than that in the control samples. Significantly, higher quantities of haemoglobin subunits were seen in MPs from malaria samples and culture supernatant compared to control samples.

Conclusion

A great number of proteins were observed to be carried in the microparticles (MPs) from malaria samples and culture supernatant compared to controls. The greater loss of haemoglobin from erythrocytes via MPs from malaria patients could serve as the initiation and progression of anaemia in P.falciparum infection. Also while some proteins were upregulated in circulating MPs in malaria samples, others were down regulated.

     

The Plasmodium knowlesi MAHRP2 ortholog localizes to structures connecting Sinton Mulligan's clefts in the infected erythrocyte

During development within the host erythrocyte malaria parasites generate nascent membranous structures which serve as a pathway for parasite protein transport to modify the host cell. The molecular basis of such membranous structures is not well understood, particularly for malaria parasites other than Plasmodium falciparum. To characterize the structural basis of protein trafficking in the Plasmodium knowlesi-infected erythrocyte, we identified a P. knowlesi ortholog of MAHRP2, a marker of the tether structure that connects membranous structures in the P. falciparum-infected erythrocyte. We show that PkMAHRP2 localizes on amorphous structures that connect Sinton Mulligan's clefts (SMC) to each other and to the erythrocyte membrane. Three dimensional reconstruction of the P. knowlesi-infected erythrocyte revealed that the SMC is a plate-like structure with swollen ends, reminiscent of the morphology of the Golgi apparatus. The PkMAHRP2-localized amorphous structures are possibly functionally equivalent to P. falciparum tether structure. These findings suggest a conservation in the ultrastructure of protein trafficking between P. falciparum and P. knowlesi.