Fig. 5: H. pylori-induced DNA damage and replication stress is prevented by overexpression of RNase H1.

a, b U2OS cells were either infected for 6 h with H. pylori P12 (MOI of 20 or 50), or treated with 100 nM camptothecin (CPT), and were treated or not with doxycycline (−/+ DOX) to induce the expression of RNase H1. Cells were subjected to immunofluorescence staining for 53BP1 and PCNA as well as DAPI. Representative images are shown in a alongside scatter dot plots of >1382 and up to 1752 cells per condition in b. Data in b are pooled from three independent experiments. c, d U2OS cells were infected as described in a, b and additionally labeled sequentially with CIdU and IdU as shown in Fig. 4a for the assessment of replication tract length. Representative DNA fibers are shown in c and scatter dot plots of CIdU tract length (in μm) are shown in d for the indicated conditions. At least 100 and up to 500 fibers were analyzed per condition. Data in d are pooled from three independent experiments. e, f U2OS cells were exposed to α- or β-ADP-heptose at 0.5 μM final concentration for 6 h and treated or not with doxycycline (−/+ DOX) to induce the expression of RNase H1. Cells were subjected to immunofluorescence staining for 53BP1 and PCNA as well as DAPI. Representative images are shown in e alongside scatter dot plots of >1500 and up to 1752 cells per condition in f. Data in f are pooled from three independent experiments. Scale bar in a and e, 10 μm. g, h U2OS cells were exposed to α- or β-ADP-heptose at 0.5 μM final concentration for 6 h, treated or not with doxycycline (−/+ DOX) to induce the expression of RNase H1, and additionally labeled sequentially with CIdU and IdU for the assessment of DNA fiber tract length. Representative DNA fibers are shown in g and scatter dot plots of CIdU tract length (in μm) are shown in h for the indicated conditions. At least 299 and up to 514 fibers were analyzed per condition. Data in h are pooled from three independent experiments. Red lines indicate medians throughout. In b, d, f, and h, −DOX and +DOX samples of the same condition are color-coded to facilitate comparisons. P-values were calculated by one-way ANOVA with Dunn’s multiple comparisons correction. ns, not significant; **p < 0.01, ***p < 0.005, ****p < 0.0001.