The integrity of the eukaryotic cellular genome is preserved by surveillance mechanisms that coordinate DNA replication, repair, and recombination with cell-cycle progression (Muzi-Falconi et al., 2003; Lazzaro et al., 2009
). The DNA nature of the chromosomes provides for an intrinsic stability as opposed to the fragility of RNA, which is due to the higher reactivity of ribose compared to deoxyribose. The incorporation of ribonucleotides (rNTPs) in place of deoxyribonucleotides (dNTPs) within genomic DNA is generally avoided by the high selectivity of DNA polymerases, largely due to a steric gate residue in the polymerase active site (Joyce, 1997
). However, there are occasions when rNTPs can be linked to DNA chains, such as during the synthesis of Okazaki fragments or possibly during repair of double strand DNA breaks in G1 (Nick McElhinny and Ramsden, 2003; Zhu and Shuman, 2008
). Recent work indicates that during normal DNA replication, DNA polymerases can also incorporate rNTPs in place of dNTPs (Nick McElhinny et al., 2010b
). rNMPs embedded in DNA are expected to represent a problem for cycling cells, sensitizing the DNA backbone to spontaneous and/or enzymatic nicking. Indeed, the presence of rNMPs in the yeast genome elevates the rate of short deletions in repeated sequences through a mechanism depending on topoisomerase I (Nick McElhinny et al., 2010a; Clark et al., 2011; Kim et al., 2011
). Furthermore, the presence of rNMPs alters DNA helix parameters. For example, structural studies (Egli et al., 1993; Jaishree et al., 1993; Ban et al., 1994a; Ban et al., 1994b; Wahl and Sundaralingam, 2000
) indicate that rNMPs in dsDNA alter global conformation from B- to A-form, with most of the sugars adopting C3′-endo or closely related conformations. rNMPs must be removed prior to the next cell cycle or they will pose problems during subsequent rounds of replication; in fact, efficient and accurate synthesis by replicative DNA polymerases strongly depends on helix geometry, such that changes in sugar pucker could render a primer terminus more difficult to extend. Indeed, a recent study has shown that single rNMPs in DNA templates impede DNA synthesis by the yeast replicases (Watt et al., 2011
). Altered helix geometry may be less problematic for polymerases specialized for translesion synthesis, e.g., DNA polymerase ζ, which can efficiently extend aberrant primer termini (Prakash et al., 2005
). An important question is thus how cells cope with replicating chromosomes containing rNMPs that escape repair.
RNase H is a family of enzymes that cleave the RNA moiety in RNA:DNA hybrids, allowing the reconstruction of a dsDNA molecule. Eukaryotic cells possess RNase H1 and RNase H2 activities that have partially overlapping substrate specificity. While RNase H1 requires at least a tract of four rNMPs to cleave, RNase H2 can incise 5′ to a single rNMP incorporated within a DNA molecule (Cerritelli and Crouch, 2009
). The in vivo roles of RNase H in eukaryotic cells are still not fully understood. In mammalian cells, RNase H1 is essential for mitochondrial DNA replication (Cerritelli et al., 2003
); such function is not conserved in budding yeast cells. The role of the nuclear population of RNase H1 is still not clear. RNase H2 represents the major RNase H activity in eukaryotic cells and is involved in several cellular processes (Cerritelli and Crouch, 2009
). Evidence indicates that these enzymes can process Okazaki fragments during replication although, at least in budding yeast, such activity is redundant and Okazaki fragment processing can be carried out by Rad27 and Dna2 (Rydberg and Game, 2002; Ayyagari et al., 2003
). Furthermore, removal of R-loops, which accumulate when a transcription bubble collides with a replication fork, can be achieved by overexpressing RNase H (Huertas and Aguilera, 2003
). Mutations in any of the three subunits of human RNase H2 are the molecular cause of a human genetic syndrome known as Aicardi-Goutières syndrome (AGS) (Crow et al., 2006a
). The mechanism(s) involved in the pathogenesis of AGS is under intense investigation but still uncertain (Crow et al., 2006b; Yang et al., 2007; Stetson et al., 2008; Rice et al., 2009; Crow and Rehwinkel, 2009
Another enzyme that processes rNMPs in DNA is topoisomerase I. It was recently reported that, in the absence of RNase H2, rNTPs incorporated in DNA are targeted by topoisomerase I, which cleaves but fails to rejoin the DNA backbone, generating a ssDNA break (Sekiguchi and Shuman, 1997; Kim et al., 2011
). Interestingly, not all genomic rNMPs are topoisomerase I targets (Kim et al., 2011
), and cells lacking RNase H2 do not exhibit growth defects, suggesting that cells must have other pathways allowing them to replicate rNMP-containing chromosomes.
In this work, we investigate the processes permitting yeast cells to survive in the presence of elevated rNTPs incorporated within genomic DNA. We show that both RNase H1 and RNase H2 play a critical role in repairing rNMPs incorporated by replicative polymerases, and in the absence of RNase H activity residual genomic rNMPs cause replication problems in the following cell cycle. When the replicative DNA polymerases encounter rNMPs in the template strand, endogenous replication stress is generated, which sensitizes cells to mild treatments with exogenous replication stress-inducing agents. In this situation, postreplication repair mechanisms are effectively responsible for the survival of RNase H defective cells. We provide genetic and biochemical evidence that rNMPs-containing chromosomes can be fully replicated through the action of template switch and DNA polymerase ζ, which efficiently bypasses rNMPs in a DNA template.
Our data show unexpected mechanisms that preserve genome integrity in normally replicating cells, extend the role of PRR, and particularly that of Pol ζ, to the replication of rNMPs in genomic DNA, and reveal a synthetic interaction between PRR, RNase H activities, and replication stress that may have relevant consequences for human disease, identifying a possible family of modifier genes that may influence the penetrance of a set of AGS mutations.