Abstract
SUMMARYRNase H2 has two distinct functions: initiation of the ribonucleotide excision repair (RER) pathway by cleaving ribonucleotides (rNMPs) incorporated during DNA replication and processing the RNA portion of an R-loop formed during transcription. An RNase H2 mutant lacking RER activity but supporting R-loop removal revealed that rNMPs in DNA initiate p53-dependent DNA damage response and early embryonic arrest in mouse. However, an RNase H2 AGS-related mutant with residual RER activity develops to birth. Estimations of the number of rNMPs in DNA in these two mutants define a ribonucleotide threshold above which p53 induces apoptosis. Below the threshold, rNMPs in DNA trigger an innate immune response. Compound heterozygous cells, containing both defective enzymes, retain rNMPs above the threshold, indicative of competition for RER substrates between active and inactive enzymes, suggesting that patients with compound heterozygous mutations in RNASEH2 genes may not reflect the properties of recombinantly expressed proteins.
Highlights
Errors during DNA replication and repair processes can challenge genome integrity
The highly efficient ribonucleotide excision repair (RER) pathway is initiated by incision at the ribonucleotide monophosphates (rNMPs) by RNase H2 followed by repair synthesis to replace rNMPs by deoxyribonucleotide monophosphates (dNMPs) (Sparks et al, 2012)
We found that Rnaseh2aG37S/À mice survived to birth, they became embryonic lethal at E12.5 in Rnaseh2aG37S/RED mice, suggesting that the RNase H2RED enzyme acts in a dominant manner for limiting the RER pathway
Summary
Errors during DNA replication and repair processes can challenge genome integrity. Cells are well equipped to restore normal duplex DNA, but in some circumstances defects may persist, leading to severe DNA damage. Discrimination against rNTPs by DNA polymerases at the sugar moiety is far from perfect, and the three replicative DNA polymerases a, d, and ε include ribonucleotide monophosphates (rNMPs) at different frequencies, resulting in about 1 rNMP for every 7,000 deoxyribonucleotide monophosphates (dNMPs) in DNA in mouse (Hiller et al, 2012; Reijns et al, 2012) and yeast (Lujan et al, 2012; Williams et al, 2016) cells. Complete absence of RNase H2 eliminates both activities, making it difficult to assign any phenotype to defects in RER or RNA/DNA hydrolysis. We modified the Rnh201 catalytic subunit in yeast RNase H2 to retain RNA/DNA activity, eliminating almost completely its rNMP cleavage activity, resulting in an RNase H2RED (ribonucleotide excision defective) (Chon et al, 2013). The RNA/DNA hybrid activity of RNase H2RED rescued the growth defect of top1Drnh1Drnh201D strain and the synthetic slow growth of sgs1Drnh201D strain, both indicative of defects in RNA/DNA processing
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