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

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

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

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

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

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