Mismatch repair in T. brucei: roles in protection against oxidative damage

Cells are continuously exposed to different intracellular and extracellular mutagens, which can damage several different molecules, including DNA. To ensure survival, cells have evolved various defence and repair mechanisms. Mismatch repair (MMR) is the mechanism that serves to repair mismatched bases in DNA that are missed by the proof reading activity of DNA polymerases. Besides this, MMR also corrects base mismatches formed by altered bases modified by certain chemical mutagens. Thus, MMR is important to avoid mutagenesis and maintain genome fidelity. MMR is a complex, highly conserved pathway that involves a range of proteins along with several accessory proteins. In T. brucei, as in other eukaryotes, MMR core functions are carried out by bacterial MutS and MutL homologues, working as heterodimers: MSH2α (MSH2-MSH3) and MSH2β (MSH2-MSH6), and MLH1-PMS1, respectively. To date, only MSH2 and MLH1 function have been examined and only in bloodstream form (BSF) T. brucei cells. It was observed that MMR mutants are tolerant towards methylation damage, exhibit microsatellite instability and display elevated rates of homologous recombination between imperfectly matched DNA molecules. This confirmed the role of MMR in genome maintenance in BSF T. brucei. More recently, it was observed that BSF MSH2 mutants were sensitive towards oxidative damage, though the same phenotype was not observed in MLH1 mutants. This suggests that some aspect of the MMR machinery acts to protect BSF T. brucei cells against oxidative stress, but the machinery and mode of action is unknown. In this study we have generated null mutants of MSH2 and MLH1 in procyclic form (PCF) T. brucei cells, and MSH3 and MSH6 mutants in BSF cells. Characterization of tolerance to DNA methylation damage and evaluating microsatellite stability shows that each gene acts in MMR in both the life cycle stages, with the exception of MSH3, where null mutants show no discernible phenotypes. Mutants were also analyzed for their action towards oxidative stress in both the life stages and, remarkably, we find life cycle stage differences, with MSH2 mutants displaying hydrogen peroxide sensitivity and resistance in the BSF and PCF, respectively. The same phenotypes are not seen in MLH1 mutants, and we show that resistance to hydrogen peroxide in PCF cells is due to adaptation during the loss of MSH2. We have also shown that PCF MSH2 mutants may show a decrease in microsatellite instability when subjected to oxidative stress. This leads to the hypothesis that there might be an unidentified system, apart from MMR, present in T. brucei PCF cells that works as a defence in response to oxidative stress and can assume greater prominence when MSH2 is lost. Although we have tried to explore various cellular processes that might contribute this activity, our results are inconclusive. MSH2 and MLH1 have also been epitope tagged to explore the subcellular localization of these proteins and to ask if any changes in expression levels or changes in localisation are seen when subjected to oxidative stress. These preliminary data suggest that both factors are nuclear and cytoplasmic. We have also tried to ask if MSH2 and/or MLH1 co-localize with either MSH5 or MSH4, which are MutS-like factors that act in meiosis in other eukaryotes, but whose functions have not been explored in T. brucei. However, our attempts at this analysis have been unsuccessful

Trypanosoma brucei and Trypanosoma cruzi DNA mismatch repair proteins act differently in the response to DNA damage caused by oxidative stress

MSH2, associated with MSH3 or MSH6, is a central component of the eukaryotic DNA Mismatch Repair (MMR) pathway responsible for the recognition and correction of base mismatches that occur during DNA replication and recombination. Previous studies have shown that MSH2 plays an additional DNA repair role in response to oxidative damage in Trypanosoma cruzi and Trypanosoma brucei. By performing co-immunoprecipitation followed by mass spectrometry with parasites expressing tagged proteins, we confirmed that the parasites’ MSH2 forms complexes with MSH3 and MSH6. To investigate the involvement of these two other MMR components in the oxidative stress response, we generated knockout mutants of MSH6 and MSH3 in T. brucei bloodstream forms and MSH6 mutants in T. cruzi epimastigotes. Differently from the phenotype observed with T. cruzi MSH2 knockout epimastigotes, loss of one or two alleles of T. cruzi msh6 resulted in increased susceptibility to H2O2 exposure, besides impaired MMR. In contrast, T. brucei msh6 or msh3 null mutants displayed increased tolerance to MNNG treatment, indicating that MMR is affected, but no difference in the response to H2O2 treatment when compared to wild type cells. Taken together, our results suggest that, while T. cruzi MSH6 and MSH2 are involved with the oxidative stress response in addition to their role as components of the MMR, the DNA repair pathway that deals with oxidative stress damage operates differently in T. brucei

High Genetic Diversity in the Himalayan Common Bean (Phaseolus vulgaris) Germplasm with Divergence from Its Center of Origin in the Mesoamerica and Andes

The common bean is found in the Himalayan region of Pakistan with substantial morphological variability. Genetic diversity within any crop species is a precursor for genetic improvement; however, little is known about common bean genetic diversity in this region. We explored the genetic diversity in the common bean from the Himalayan region (Khyber Pakhtunkhwa, Gilgit–Baltistan, Kashmir) of Pakistan. Microsatellite genotyping was carried out for 147 samples with 40 simple sequence repeat (SSR) markers. The results revealed a clear divergence of the Pakistani population from the primary gene pool (with FST values of 0.2 with Andes and 0.27 with Mesoamerica). However, within the Himalayan germplasm, no clear evidence of spatial structure was observed (with the maximum FST values of only 0.025), probably due to the dispersal of seeds by human activity within the region. This was further elucidated by the discriminant analyses of principal components. Considering the diversity parameters, high genotypic diversity was observed for the indigenous lines (0.990), comparable to the primary gene pool (0.976 for Mesoamerica and 0.976 for Andes populations). A high genotypic diversity was observed within the Himalayan population (ranging from 0.500 for Upper Dir to 0.952 for Mansehra). Gene diversity across loci varied between 0.28 for Chitral to 0.38 for Kurram. Our results suggested a divergent and independent evolution of the Himalayan population, which might have led to the diversification of the common bean germplasm in the region postintroduction into the region. The diversity observed could also be exploited in future breeding programs for the development and introduction of climate-resilient varieties

Re-expression of MSH2 in <i>T</i>. <i>brucei msh2</i> null mutants.

<p>(<b>A</b>) Procyclic form (PCF) <i>T</i>. <i>brucei</i> (Tb) wild type (WT), <i>Tbmsh2</i>+/-, <i>Tbmsh2</i>-/- and <i>Tbmsh2</i>-/- cells in which MSH2 is re-expressed (<i>Tbmsh2</i>-/-/+) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated. (<b>B</b>) PCF WT, <i>Tbmsh2</i>+/-, <i>Tbmsh2</i>-/- and <i>Tbmsh2</i>-/-/+ cells were grown in culture medium with 0 μM, 10 μM or 20 μM H₂O₂ and cell density was determined 48 and 72 hours later; growth is shown percentage survival of the treated cells relative to untreated (<b>C</b>) Growth of wild type bloodstream form (BSF) <i>T</i>. <i>brucei</i> cells was compared to <i>Tbmsh2</i>+/-, <i>Tbmsh2</i>-/- and <i>Tbmsh2</i>-/-/+ BSF mutants in the presence of 100 μM or 200 μM H₂O₂ as described above; graph shows survival of the mutants after 48 hours growth plotting the density of the treated cells as a percentage of the untreated; vertical lines show standard deviation. ***p<0.001, **p<0.001: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type; ns indicates no significant difference.</p

8-oxoguanine (8-oxoG) accumulation in MSH2 knockout cells.

<p>FITC-avidin was used to estimate 8-oxoG levels based on the fluorescence intensity in the nuclear DNA (nDNA) and kinetoplast DNA (kDNA) of <i>T</i>. <i>brucei</i> procyclic forms and <i>T</i>. <i>cruzi</i> epimastigotes. (<b>A</b>) Representative images of FITC-avidin or DAPI stained <i>T</i>. <i>brucei</i> WT cells and <i>Tbmsh2</i>+/- or <i>Tbmsh2</i>-/- mutants are shown. (Bar = 1.9 μM) (<b>B</b>) Fluorescence intensity of FITC-avidin signals were quantified in the nDNA and kDNA using ImageJ software and plotted as arbitrary units; values shown are the average signal from 100 WT, <i>Tbmsh2+/-</i>, <i>Tbmsh2-/-</i>, <i>Tbmlh1+/-</i> or <i>Tbmlh1-/-</i> PCF cells; vertical lines show standard error (SEM). (<b>C</b>) FITC-avidin signal evaluated by the same process in <i>T</i>. <i>cruzi</i> epimastigote WT cells and in <i>msh2+/-</i> and <i>msh2-/-</i> knockout mutants. ***p<0.001: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type; ns indicates no signifcant difference.</p

Generation of mismatch repair null mutants in <i>T</i>. <i>cruzi</i> and <i>T</i>. <i>brucei</i>.

<p>(<b>A</b>) Agarose gel electrophoresis of reverse transcriptase (RT) PCR products to verify the absence of <i>MSH2</i> or <i>MLH1</i> mRNA in <i>T</i>. <i>brucei</i> procyclic form mutants. cDNA derived from wild type (WT) cells, blasticidin (BSD) or puromycin (PUR) resistant clones of <i>Tbmsh2</i> single allele knockouts (+/-) and <i>Tbmsh2</i> double allele knockouts (-/-) were PCR-amplified with <i>MSH2</i> or <i>MLH1</i>- specific primers; the size of the PCR product is indicated and a control reaction without cDNA is indicated by C. The bottom panel shows, as positive controls, cDNAs from the same cells PCR-amplified (RT+) with primers specific for the <i>RAD51</i> gene; a control for genomic DNA contamination, in which reverse transcriptase was excluded from the cDNA synthesis reaction (indicated by RT-) is also shown. (<b>B</b>) Total RNA extracted from <i>T</i>. <i>cruzi</i> epimastigote form wild type (WT) cells, a <i>Tcmsh2</i> single allele knockout (+/-) mutant and three independent clones of <i>Tcmsh2</i> double allele knockouts (-/-) were transferred to a nylon membrane and hybridized with [α-³²P]-labeled probe specific for the <i>T</i>. <i>cruzi MSH2</i> gene. The bottom panel shows hybridization with a probe for 24Sα rRNA, used as loading control.</p

Assessment of <i>T</i>. <i>cruzi msh2</i> knockout mutant infectivity <i>in vitro</i>.

<p>(<b>A</b>) <i>T</i>. <i>cruzi</i> trypomastigote cells released by Vero cells infected with either WT or with three cloned cell lines of <i>Tcmsh2</i>-/- mutants were counted and equal numbers were used to infect Vero cells attached to glass coverslips or (<b>B</b>) cultured intraperitoneal macrophages extracted from BALB/c mice. Graphs show the average number of intracellular parasites counted per 100 cells; vertical lines denote standard deviation. **p<0.01, *p<0.05: one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type.</p

Susceptibility of <i>T</i>. <i>brucei</i> and <i>T</i>. <i>cruzi</i> MMR knockout mutants to N-methyl-N’-nitro-N-nitrosoguanidine (MNNG).

<p>(<b>A</b>) <i>T</i>. <i>brucei</i> wild type (WT) and procyclic form mutants (<i>Tbmsh2</i>+/-, <i>Tbmsh2</i>-/-, <i>Tbmlh1</i>+/- and <i>Tbmlh1</i>-/-) were grown in culture medium with 0 μM, 2.5 μM or 5 μM MNNG. Cell density was measured after 72 hours growth and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. (<b>B</b>) WT <i>T</i>. <i>cruzi</i> epimastigotes and MSH2 mutants (<i>Tcmsh2</i>+/- and <i>msh2</i>-/-) were grown in culture medium with 0 μM or 5 μM MNNG. Cell viability was measured after 72 hours and is plotted as the percentage survival of the MNNG treated cells relative to untreated cultures. Vertical lines indicate standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of knockout mutants relative to wild type cells.</p

MSH2 knockout mutants are more resistant to oxidative stress generated by H₂O₂.

<p>(<b>A</b>) <i>T</i>. <i>brucei</i> wild type (WT), <i>msh2</i>+/-, <i>msh2</i>-/-, <i>mlh1</i>+/- and <i>mlh1</i>-/- procyclic form cells were grown in culture medium with 0 μM, 10 μM or 20 μM H₂O₂. Cell density was measured after 48 hours and plotted as the percentage survival of the H₂O₂ treated cells relative to untreated. (<b>B</b>) <i>T</i>. <i>cruzi</i> epimastigote WT, <i>msh2</i>+/- and <i>msh2</i>-/- cells were incubated with or without 75 μM H₂O₂ for 20 minutes in PBS 1x and then allowed to grow in LIT medium for 48 hours, after which cell viability was determined and plotted as percentage survival of the treated cells relative to untreated. Vertical lines show standard deviation. ***p<0.001, **p<0.01, *p<0.05: determined by one-way ANOVA with Bonferroni post-test of mutants relative to wild type cells; ns indicates no signifcant difference.</p