ED Figure 4.
Related to Figure 2j. RNASEH2A P40D/Y210A is a separation-of-function mutant that cannot excise single DNA-embedded ribonucleotides, but cleaves RNA:DNA heteroduplexes (similar to the yeast rnh201-P45D-Y219A mutant16). a, Schematic depicting enzymatic activity against two different RNase H2 substrates (DRD:DNA, dsDNA with embedded ribonucleotide, or RNA:DNA hybrids) in cell lines used in b-d and Fig 2j. WT and RNASEH2A-KO cells were transduced with either an empty vector (EV) or the indicated RNASEH2A constructs. b, Complementation of HeLa RNASEH2A-KO cells with FLAG-tagged RNASEH2A variants restores RNase H2 complex protein levels. WCEs from HeLa WT and RNASEH2A-KO cells stably expressing indicated lentiviral constructs were processed for immunoblotting with the indicated antibodies. Vinculin, loading control. Asterisk indicates a non-specific band. Representative of n = 3 biologically independent experiments. c,d, Complementation of HeLa RNASEH2A-KO cells with WT RNASEH2A, but not with the D34A/D169A (catalytic-dead) or P40D/Y210A (separation-of-function) mutants, rescues increased levels of genome-embedded ribonucleotides. c, Total nucleic acids from the cell lines shown in a, b were treated with recombinant RNase H2 and separated by alkaline agarose gel electrophoresis (representative of n = 4 experiments). d, Densitometric quantification of alkaline gel shown in c. e, Purified human RNase H2 complexes consisting of RNASEH2B, RNASEH2C and either RNASEH2A WT, P40D/Y210A or D34A/D169A subunits separated by SDS-PAGE and stained with Coomassie Blue (n = 1). f-k, RNase H2 activity assays with fluorescein-labeled RNA:DNA substrate (f) or double-stranded DNA with a single incorporated ribonucleotide (DRD:DNA) (g) and increasing amounts of recombinant WT, P40D/Y210A or D34A/D169A RNase H2. Products were separated by polyacrylamide gel electrophoresis and detected by fluorescence imaging. Representative of n = 3 biologically independent experiments. h,k, Quantification of f, g. Product signal plotted relative to substrate signal per lane. Mean ±SD (n = 3 biologically independent experiments).