RNase H and Postreplication Repair Protect Cells from Ribonucleotides Incorporated in DNA

Abstract

SummaryThe chemical identity and integrity of the genome is challenged by the incorporation of ribonucleoside triphosphates (rNTPs) in place of deoxyribonucleoside triphosphates (dNTPs) during replication. Misincorporation is limited by the selectivity of DNA replicases. We show that accumulation of ribonucleoside monophosphates (rNMPs) in the genome causes replication stress and has toxic consequences, particularly in the absence of RNase H1 and RNase H2, which remove rNMPs. We demonstrate that postreplication repair (PRR) pathways—MMS2-dependent template switch and Pol ζ-dependent bypass—are crucial for tolerating the presence of rNMPs in the chromosomes; indeed, we show that Pol ζ efficiently replicates over 1–4 rNMPs. Moreover, cells lacking RNase H accumulate mono- and polyubiquitylated PCNA and have a constitutively activated PRR. Our findings describe a crucial function for RNase H1, RNase H2, template switch, and translesion DNA synthesis in overcoming rNTPs misincorporated during DNA replication, and may be relevant for the pathogenesis of Aicardi-Goutières syndrome.

Structure–function analysis of ribonucleotide bypass by B family DNA replicases

“…This relatively efficient bypass is consistent with the small structural changes in L415F RB69 Pol ternary complexes bound to ribonucleotide-containing template primers (Fig. 2), as well as with the ability of ribonucleotide repair-deficient yeast (5,6,10,17) and mice (7,19) to survive and replicate genomes that contain large numbers of ribonucleotides. The fact that ribonucleotides incorporated into the genome by DNA polymerases can be tolerated is consistent with the idea that they can have beneficial signaling functions (4,9,11,12).…”

“…A second possibility for consecutive ribonucleotides in DNA that cannot be excluded by current studies in yeast (5,6,10,17) or mice (7,19) is incorporation of consecutive ribonucleotides during synthesis by DNA replicases, or perhaps by less faithful DNA polymerases. However, a third possibility for bypass of consecutive ribonucleotides was suggested by a study demonstrating that when DNA oligonucleotides containing consecutive ribonucleotides are introduced into yeast, they can direct the template-dependent repair of double strand DNA breaks (41).…”

“…achieve efficient and accurate DNA synthesis, and with crystallographic and NMR studies (21)(22)(23)(24) showing that ribonucleotides in DNA alter helix parameters. Recent studies have shown that Pols δ and e have difficulty bypassing ribonucleotides in DNA templates (4), whereas Pol ζ does not (17). The probability that Pol e will pause during single ribonucleotide bypass increases after dNTP insertion opposite the ribonucleotide and for several additional insertions opposite deoxynucleotides (5,25).…”

“…Yeast strains defective in RNase H2-dependent ribonucleotide excision repair (RER) (5,14) exhibit several characteristics of replicative stress, including strongly elevated rates for deleting 2-5 bp from repetitive DNA sequences (5,15), events that are initiated by topoisomerase 1 cleavage of a ribonucleotide in DNA (10,16). Yeast strains defective in RNase H2 and RNase H1 progress slowly through S phase, accumulate ubiquitylated proliferating cell nuclear antigen (PCNA), and are sensitive to treatment with hydroxyurea (17). Moreover, their survival in the presence of hydroxyurea partly depends on MMS2-dependent template switching and on REV3, which encodes the catalytic subunit of the translesion synthesis (TLS) enzyme Pol ζ.…”

“…Moreover, their survival in the presence of hydroxyurea partly depends on MMS2-dependent template switching and on REV3, which encodes the catalytic subunit of the translesion synthesis (TLS) enzyme Pol ζ. When ribonucleotide incorporation during leading strand replication is increased by a M644G substitution in the Pol e active site, a defect in RNase H2 results in elevated deletion mutagenesis, elevated dNTP pools, slow growth and activation of the S-phase checkpoint (5,10,18), and concomitant deletion of the RNH1 gene encoding RNases H1 is lethal (17). In mice, knocking out any of the genes encoding the three subunits of RNase H2 is embryonic lethal (7,19).…”