Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair
2019; Springer Nature; Volume: 39; Issue: 3 Linguagem: Inglês
10.15252/embj.2019102309
ISSN1460-2075
Autores Tópico(s)RNA and protein synthesis mechanisms
ResumoReview12 December 2019Open Access Molecular and physiological consequences of faulty eukaryotic ribonucleotide excision repair Vanessa Kellner Corresponding Author Vanessa Kellner [email protected] orcid.org/0000-0002-7060-2520 Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Brian Luke Corresponding Author Brian Luke [email protected] orcid.org/0000-0002-1648-5511 Institute of Molecular Biology (IMB), Mainz, Germany Institute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg Universität, Mainz, Germany Search for more papers by this author Vanessa Kellner Corresponding Author Vanessa Kellner [email protected] orcid.org/0000-0002-7060-2520 Institute of Molecular Biology (IMB), Mainz, Germany Search for more papers by this author Brian Luke Corresponding Author Brian Luke [email protected] orcid.org/0000-0002-1648-5511 Institute of Molecular Biology (IMB), Mainz, Germany Institute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg Universität, Mainz, Germany Search for more papers by this author Author Information Vanessa Kellner *,1,3 and Brian Luke *,1,2 1Institute of Molecular Biology (IMB), Mainz, Germany 2Institute of Developmental Biology and Neurobiology (IDN), Johannes Gutenberg Universität, Mainz, Germany 3Present address: Department of Biology, New York University, New York, NY, USA *Corresponding author. Tel: +1 212 992 6590; E-mail: [email protected] *Corresponding author. Tel: +49 6131 39 21465; E-mail: [email protected] The EMBO Journal (2020)39:e102309https://doi.org/10.15252/embj.2019102309 See the Glossary for abbreviations used in this article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The duplication of the eukaryotic genome is an intricate process that has to be tightly safe-guarded. One of the most frequently occurring errors during DNA synthesis is the mis-insertion of a ribonucleotide instead of a deoxyribonucleotide. Ribonucleotide excision repair (RER) is initiated by RNase H2 and results in error-free removal of such mis-incorporated ribonucleotides. If left unrepaired, DNA-embedded ribonucleotides result in a variety of alterations within chromosomal DNA, which ultimately lead to genome instability. Here, we review how genomic ribonucleotides lead to chromosomal aberrations and discuss how the tight regulation of RER timing may be important for preventing unwanted DNA damage. We describe the structural impact of unrepaired ribonucleotides on DNA and chromatin and comment on the potential consequences for cellular fitness. In the context of the molecular mechanisms associated with faulty RER, we have placed an emphasis on how and why increased levels of genomic ribonucleotides are associated with severe autoimmune syndromes, neuropathology, and cancer. In addition, we discuss therapeutic directions that could be followed for pathologies associated with defective removal of ribonucleotides from double-stranded DNA. Glossary AGS Aicardi–Goutières syndrome AOA1 Ataxia with oculomotor apraxia 1 dNTP deoxyribonucleotide triphosphate DSB double-strand break GCR gross chromosomal rearrangements HDR homology-directed repair HR homologous recombination INF interferon LOH loss of heterozygosity MMR mismatch repair NER nucleotide excision repair NHEJ nonhomologous end-joining PARP (poly-ADP)ribose polymerase PIP-box PCNA-interacting motif RER ribonucleotide excision repair RNAPII RNA polymerase II RNase H ribonuclease H rNMP ribonucleoside monophosphate rNTP ribonucleotide triphosphate SLE systemic lupus erythematosus SSB single-strand break ssDNA single-stranded DNA Top1cc topoisomerase 1 cleavage complex Top1 topoisomerase 1 Introduction It is of critical importance that the duplication of human genomes is a tightly regulated and safe-guarded process. The insertion of erroneous bases by replicative DNA polymerases can potentially manifest into heritable mutations with pathological consequences. Considerable emphasis has been placed on understanding the causes, consequences, and repair of faulty base incorporation, which leads to DNA mismatches. In some instances, the replicative polymerases still get it wrong despite the correct base being inserted and a Watson–Crick basepair being formed; this can occur when the inappropriate sugar moiety attached to the base is selected. The replication of DNA utilizes deoxyribonucleotide triphosphates (dNTPs) to faithfully duplicate the genome. The synthesis of RNA via transcription, on the other hand, employs ribonucleotide triphosphates (rNTP), where the 2′-carbon atom of the ribose sugar is hydroxylated. Intracellular concentrations of rNTPs highly exceed those of dNTPs (between 30- and 200-fold in budding yeast depending on the base) (Nick McElhinny et al, 2010b), thereby increasing the likelihood that rNTPs are mistakenly used instead of dNTPs, despite the presence of the correct base attached. The three replicative DNA polymerases (Pol α, Pol ε, and Pol δ) harbor a highly conserved tyrosine residue adjacent to the active polymerization site that acts as “steric gate” to limit rNTP incorporation during DNA replication, by excluding the hydroxy group of the ribose (Brown & Suo, 2011). rNTP exclusion via the steric gate is however not flawless, and in vitro studies using endogenous concentrations of rNTPs and dNTPs suggest that in the Saccharomyces cerevisiae genome, approximately 13,000 ribonucleotides become incorporated into newly replicated DNA within a single round of replication (Nick McElhinny et al, 2010b). Similar frequencies of rNTP incorporation have been confirmed in vivo, resulting in an incorporation rate of approximately one rNMP per 6,500 bases (Lujan et al, 2013). In addition to rNTP mis-incorporation, Pol α/Primase synthesizes short RNA primers to initiate Okazaki fragments that transiently make up ~ 5% of the nascent lagging strand (Zheng & Shen, 2011). Accordingly, in human cells the number of incorporated ribonucleotides per cell cycle is estimated to reach more than one million (Clausen et al, 2013). This makes ribonucleotides the most frequently incorporated non-canonical nucleotide in duplex DNA, exceeding the combined total number of all abasic, oxidized, and otherwise modified nucleotides (Caldecott, 2014). Once the rNTP is incorporated into the context of DNA, it exists as a ribonucleoside monophosphate (rNMP). The presence of rNMPs in a DNA template directly affects the processivity of DNA polymerization during semi-conservative replication. Although budding yeast replicative polymerases can bypass a single rNMP, the in vitro efficiency of this bypass is strongly reduced (for Pol ε only about 66% bypass is achieved) and drops dramatically when replication has to transverse stretches of three or more consecutive rNMPs (Watt et al, 2011). As of yet, there is no direct in vivo evidence that DNA polymerases are affected by stretches of multiple rNMPs. Comparable in vitro observations have been made for the human replicative polymerases, Pol ε and Pol δ (Göksenin et al, 2012; Clausen et al, 2013). Therefore, the accumulation of rNMPs in genomic DNA likely induces replication stress and DNA damage signaling from yeast to human (Nick McElhinny et al, 2010a; Hiller et al, 2012; Lazzaro et al, 2012; Williams et al, 2013; Pizzi et al, 2015; Zimmermann et al, 2018) (discussed below). Thus, to preserve genome stability, rNMPs have to be efficiently removed from the genome. A dedicated repair pathway, referred to as ribonucleotide excision repair (RER), employs specialized enzymes to eliminate rNMPs. Error-free and error-prone removal of ribonucleotides DNA polymerases have a built-in proofreading mechanism whereby faulty base incorporation is corrected through exonuclease-mediated removal of the incorrect base (Burgers & Kunkel, 2017). Recognition and excision of rNMPs by DNA polymerases is only one-third as effective as their proofreading of incorrect dNMP base pairing and in the case of Pol ε has been found to most likely not significantly contribute to rNMP removal (Shcherbakova et al, 2003; Williams et al, 2012). Instead, RNase H enzymes have the capacity to cleave DNA at sites of rNMP incorporation and thus to initiate rNMP removal. RNase H is conserved in prokaryotes, but we will not cover bacterial RNase H enzymes here and instead refer interested readers to a study by Kochiwa et al (2007) for an overview on them. The eukaryotic RNase H family consists of the monomeric RNase H1 enzymes and the trimeric RNase H2 enzymes, both of which eliminate RNA-DNA hybrid structures occurring throughout the genome (Cerritelli & Crouch, 2009). While the accidentally incorporated rNMPs are found both as single bases and in longer consecutive stretches, RNA–DNA hybrids can also form when single-stranded RNA molecules anneal to a complementary DNA strand, thereby displacing the second strand of the DNA double helix. This three-stranded structure, termed R-loop, can have detrimental consequences on genome stability if not removed in a timely manner (Santos-Pereira & Aguilera, 2015). RNase H1 requires at least four consecutive rNMPs to recognize an RNA–DNA hybrid structure, a situation that occurs in the context of an R-loop. RNase H2, on the other hand, can act both on rNMP stretches such as those found in R-loops, as well as on single and consecutive rNMPs in the context of double-stranded DNA, making it a more versatile enzyme. Consistently, RNase H2 activity accounts for the bulk of RNase H activity in the cell (Sparks et al, 2012). Given the enzymatic capabilities of RNase H2, one may expect the existence of tight regulatory mechanisms in order to prevent chromosomal nicking at inappropriate times, e.g., during DNA replication. In S. cerevisiae, the trimeric RNase H2 enzyme consists of the catalytic subunit Rnh201 and the accessory subunits Rnh202 and Rnh203. Human RNase H2 shows strong conservation and comprises RNASEH2A, the catalytic subunit, as well as RNASEH2B and RNASEH2C (Crow et al, 2006). Loss of any of its subunits renders the enzyme complex inactive (Jeong et al, 2004). The dual activity of RNase H2 toward R-loops and rNMPs can be largely, but not entirely, uncoupled by the use of a separation-of-function allele of the catalytic Rnh201 subunit, RNH201-P45D-Y219A (or RNH201-RED for ribonucleotide excision defective). This point mutation within the substrate-interacting pocket completely abolishes activity of RNase H2 toward single rNMPs but retains approximately 40% of wildtype enzymatic activity toward longer rNMP stretches and R-loops (Chon et al, 2013). Although this allele was originally constructed in yeast, it has since been recapitulated in human and mouse cells, resulting in a similar separation-of-function phenotype (Pizzi et al, 2015; Uehara et al, 2018; Zimmermann et al, 2018). RNase H2 promotes error-free RER RNase H2 is responsible for the primary pathway of rNMP removal from genomic DNA, i.e., error-free RER. Upon recognizing an rNMP in the context of duplex DNA, RNase H2 incises the DNA backbone on the 5′ side of the ribonucleotide to allow its subsequent removal and repair (Eder et al, 1993; Rydberg & Game, 2002; Fig 1). In vitro reconstitution experiments have elucidated the RER mechanism in detail (Sparks et al, 2012): Initial incision at the DNA–RNA junction mediated by RNase H2 produces a single-stranded DNA break flanked by a 3′ hydroxy (3′OH) group and a 5′ phosphate. Starting from the 3′OH, Pol δ or (less efficiently) Pol ε perform strand displacement DNA synthesis, thereby creating a flap structure harboring the rNMP (Fig 1). This flap is subsequently removed by flap endonuclease (yeast Rad27/human FEN1) or the exonuclease Exo1. Finally, the remaining single-stranded nick is sealed by DNA ligase (Sparks et al, 2012). Crystal structures of bacterial, mouse, and human RNase H2 enzymes have allowed to further dissect their substrate recognition, binding, and hydrolysis mechanisms (Rychlik et al, 2010; Shaban et al, 2010; Figiel et al, 2011): RNase H2 recognizes rNMPs at the (5′)RNA–DNA(3′) junction. The 5′-phosphate of the rNMP is positioned into the active site of the complex, while its 2′OH interacts with a glycine–arginine–glycine (GRG) motif and a conserved tyrosine residue within the catalytic subunit, thereby improving substrate selectivity (Rychlik et al, 2010). The catalytic step takes place in the active site consisting of four conserved carboxylates, which coordinate metal ions and water molecules to attack the phosphate bond 5′ of the rNMP (Rychlik et al, 2010; Shaban et al, 2010). Figure 1. Overview of ribonucleotide excision repair (RER)RNase H2 initiates RER by incising the DNA backbone at the rNMP (R in red circle). Nick translation DNA synthesis from the newly created 3′OH followed by FEN1/Exo1-mediated flap processing and subsequent DNA ligation can efficiently repair the incised DNA, resulting in removal of the rNMP. Download figure Download PowerPoint Error-prone rNMP removal by Top1 in the absence of RNase H2 Prior to characterization of the RNase H2-based RER mechanism in such intricate detail, in vitro work had demonstrated that topoisomerase 1 (Top1) can also process an rNMP-containing DNA substrate (Sekiguchi & Shuman, 1997). More recently, this Top1-dependent mechanism was also shown to remove rNMPs from DNA in vivo and thus to represent an important backup mechanism in RER-defective cells lacking RNase H2 activity (Williams et al, 2013). In this reaction, the catalytic tyrosine residue of Top1 forms a Top1 cleavage complex (Top1cc), i.e., a covalent intermediate via transesterification at the 3′ terminal phosphate of the rNMP, in a similar manner to the reaction that Top1 initiates on pure supercoiled DNA lacking rNMPs. During the latter, canonical reaction, Top1 can readily catalyze religation of the created nick; when the nick occurs at an rNMP, however, the Top1-phosphate bond is prone to attack from the adjacent 2′OH group of the ribose moiety, resulting in Top1 release and creation of an unligatable 2′,3′-cyclic phosphate (Sekiguchi & Shuman, 1997; Fig 2A). The resulting nick flanked by the 2′,3′-cyclic phosphate and a 5′OH group requires further processing before either ligation or extension is possible. Although Top1 cleavage can achieve error-free repair of rNMPs, other more detrimental repair alternatives exist (Fig 2). In fact, Top1-mediated processing of rNMPs greatly contributes to genome instability in the absence of RNase H2 (discussed below). Figure 2. Topoisomerase 1 as backup for RER in rNMP removal from the genome(A) In the absence of functional RNase H2, Top1 can act on accumulating rNMPs. Different outcomes have been characterized in budding yeast (see text for detailed descriptions), resulting either in error-free repair or in repair that causes mutations or potentially lethal double-strand breaks. (B–D) Secondary Top1-mediated incision two basepairs upstream releases an rNMP-dNMP dinucleotide and creates a Top1-linked gap (B) that can be processed in an error-free manner via Tdp1 (C), or in an error-prone manner caused by Top1 realignment and religation (D). (E) Error-free gap repair based on subsequent activities of Srs2 helicase, Exo1 exonuclease, and Apn2 abasic endonuclease, which prevent erroneous religation. (F) Secondary Top1 incision on the opposite strand creates DNA double-strand breaks that require repair by homologous recombination. Download figure Download PowerPoint Top1-mediated removal has been described to be specific for rNMPs incorporated by the leading-strand DNA polymerase Pol ε, and it appears to be resolved in different ways (Williams et al, 2013, 2015; Cho et al, 2015). In one scenario, Top1 can initiate a second cut on a dNMP two basepairs upstream of the initial cut, leading to the release of an rNMP-dNMP dinucleotide (Sparks & Burgers, 2015; Fig 2B). In this case, the covalently bound Top1cc is processed and released by tyrosyl-DNA phosphodiesterase Tdp1, leaving behind a two-nucleotide gap that can be repaired in an error-free manner (Fig 2C). As an alternative to this Tdp1-dependent pathway, and especially within tandem repeat sequences, Top1 may realign the DNA backbone and ligate the nick (Huang et al, 2015; Sparks & Burgers, 2015). In this scenario, ligation by Top1 leads to characteristic slippage mutations consisting of two- to five-basepair deletions (∆2–5 bp) (Nick McElhinny et al, 2010a; Kim et al, 2011; Fig 2D). Processing of the initial Top1-created 5′OH via Srs2 helicase and Exo1 nuclease can disfavor direct religation, thereby reducing the risk of acquiring those ∆2–5 bp slippage mutations (Potenski et al, 2014). Here, the 3′–5′ helicase activity of Srs2 unwinds the DNA from this free 5′ end, followed by flap processing via Exo1. While creation of this DNA gap reduces the likelihood of Top1-mediated ligation following a second Top1 incision, the 3′ DNA end still has to be processed to allow extension by Pol δ. Biochemical in vitro data supported by genetic interaction studies indicate that the abasic endonuclease Apn2 can process the 3′-terminal 2′,3′-cyclic phosphate and promote Pol δ extension (Li et al, 2019; Fig 2E). Yet another scenario related to yeast Top1 activity at rNMP sites has been described: Following the first incision at the rNMP, Top1 can also cut on the DNA strand opposing the rNMP to create double-strand breaks (DSBs), which then rely on homology-directed repair (HDR) via Rad51 and Rad52 (Huang et al, 2015; Fig 2F). Consistently, RNase H2 loss in human cells results in synthetic lethality with the absence of either BRCA1 or BRCA2 HDR factors, highlighting the importance of HDR under conditions of rNMP accumulation (Zimmermann et al, 2018). While rNMP removal via topoisomerase had initially only been described in S. cerevisiae, recent work employing various RER-defective RNase H2-mutant human cell lines has demonstrated that also human TOP1 can recognize an unrepaired rNMP and incise to create a DNA nick with the potential to compromise genome stability (Zimmermann et al, 2018). Whether TOP1 activity indeed removes rNMPs from human DNA as it does in yeast remains to be tested. In particular, it will be interesting to determine how Top1 recognizes rNMPs in the DNA, i.e., is Top1-mediated rNMP processing a regulated process, or rather an accidental byproduct of Top1 action in relieving supercoiling? Another outstanding question is whether or not Top1 contributes to RER in the presence of RNase H2, and if so to what extent. Moreover, what determines which Top1 repair pathway (nicking out the rNMP, nicking and re-ligating or DSB generation) will be employed at an rNMP? The latter question is particularly relevant in the context of RER-defective cells, where the different Top1-dependent processing mechanisms will have very different outcomes in terms of preserving genome integrity. Alternative removal mechanisms and tolerance of rNMPs Both yeast and mammalian cells lacking RNase H2 display signs of replication stress. In yeast mutants, the S-phase checkpoint and the postreplicative repair pathway are constitutively activated, and accordingly, cells exhibit delayed cell cycle progression (Nick McElhinny et al, 2010a; Lazzaro et al, 2012; Williams et al, 2013; Zimmermann et al, 2018). The same defects are observed in RNase H2-depleted human cells and cells from patients suffering from Aicardi–Goutières syndrome (AGS) associated with mutations in RNase H2 (discussed below) (Pizzi et al, 2015). Similarly, loss of RNase H2 in mouse cells results in altered cell cycle timing with accumulation of cells in G2/M phase, chronic activation of the DNA damage response, increase in single-strand breaks (SSBs), and increased nuclear foci harboring the phosphorylated histone variant H2AX (γH2AX) (Hiller et al, 2012). In bacteria, nucleotide excision repair (NER) can serve as backup for RER (Cai et al, 2014), but a NER contribution to rNMP removal in yeast and human cells has been largely ruled out (Lazzaro et al, 2012; Lindsey-Boltz et al, 2015). Mismatch repair (MMR), which is very efficient in recognizing and removing mismatched bases from dsDNA, also does not appear to contribute to rNMP removal in yeast (Lazzaro et al, 2012). Of note, RNase H2-mediated incision at rNMPs has conversely been assigned a guiding role during MMR: As rNMPs are transiently inserted during DNA replication, nicks subsequently created by RNase H2 serve as a guide for strand determination and ensure that the MMR machinery specifically removes mismatches on the newly synthesized strand discriminated by the nicks (Ghodgaonkar et al, 2013; Lujan et al, 2013). Therefore, RNase H2 defects may increase the mutagenic load also in an indirect manner, by decreasing the efficiency of MMR. Of note, the lethality of combined absence of both RNase H enzymes and Top1 in yeast (rnh1Δ rnh201Δ top1Δ) can be bypassed by expression of the RER-deficient, but R-loop processing-proficient RNH201-RED allele (Chon et al, 2013). This indicates that lethality in this strain is likely mediated by an accumulation of toxic R-loops and implies that yeast cells can in principle tolerate the rNMPs that accumulate in the genome when both RER and Top1-dependent repair are compromised. Therefore, either a yet unknown backup pathway for rNMP removal might exist, or yeast cells can survive high numbers of rNMPs in their genome despite a high load of DNA damage and replication stress. In cells that lack both RNase H1 and RNase H2, the postreplicative repair pathway has been found to be crucial (Lazzaro et al, 2012), but it has not been addressed to which extent this is due to either R-loops or rNMPs, leaving the involvement of this pathway in the bypass of lethality allowed by the RNH201-RED allele an interesting hypothesis to test. Another possibility to consider is that stretches of consecutive ribonucleotides, and not R-loops, are responsible for the phenotypes observed in rnh1Δ rnh201Δ cells. This would also be consistent with a genetic rescue by the RNH201-RED allele. Recent work has demonstrated that translesion polymerase eta (Pol η) can incorporate consecutive rNMP stretches at stalled replication forks in the presence of HU (Meroni et al, 2019). Consistently, the deletion of Pol η rescues the HU sensitivity of rnh1Δ rnh201Δ cells. Given the importance of RER for maintaining genome integrity, it is surprising that DNA polymerases have evolved to permit rNTP usage at all. This may suggest that, pending their timely and controlled removal, rNMPs may also exert beneficial functions in certain situations (Potenski & Klein, 2014). As discussed above, MMR on the nascent leading strand is facilitated by RNase H2-induced nicks at rNMPs (Ghodgaonkar et al, 2013; Lujan et al, 2013), and it is an intriguing possibility that other processes could be similarly affected by rNMPs incorporated by replicative DNA polymerases. Another source of rNMP incorporation with a beneficial role is in nonhomologous end-joining (NHEJ) repair, where a critical role is played by DNA polymerase mu (Pol μ), which displays even weaker sugar selectivity than replicative DNA polymerases (Potenski & Klein, 2014). Indeed, transient incorporation of rNMPs at broken DNA ends effectively enhances their subsequent ligation (Pryor et al, 2018). RER regulation Though many aspects of the RER reaction have been well-described, some rather fundamental aspects of the error-free removal of rNMPs still require further investigation. The C-terminal region of Rnh202 (hRNASEH2B) harbors a PCNA-interacting peptide motif (PIP-box) suggesting replisome association, the importance of which still remains unclear. While deletion of the PIP-box affects localization of RNase H2 to sites of PCNA-dependent DNA replication in human cells (Bubeck et al, 2011), an RNase H2 complex lacking the PIP-box (RNASEH2B-∆PIP) still retains residual co-localization with PCNA (Kind et al, 2014). In budding yeast, Rnh202-∆PIP-mutant cells grow indistinguishably from cells with wildtype Rnh202, suggesting that recognition of the rNMP rather than interaction with PCNA is crucial for RER to function (Chon et al, 2013). One possibility is that, especially in human cells, PCNA might reinforce retention of RNase H2 at sites of DNA replication or repair synthesis but is not required for its recruitment per se. This is reminiscent of the recruitment mechanism reported for DNA methyltransferase Dnmt1, where ablation of the PIP-box is compensated by direct interaction between the Dnmt1-targeting sequence and DNA (Schneider et al, 2013), or the dual recruitment of poly(ADP-ribose) glycohydrolase PARG through both its substrate poly(ADP-ribose) (PAR) and its interaction with PCNA (Mortusewicz et al, 2011). Another simple interpretation of these results would be that RNase H2 interacts with additional replisome components other than PCNA, which too may assist RNase H2 delivery to rNMPs. Alternatively, RNase H2 may recruit PCNA and other repair factors to rNMPs to promote RER, instead of the other way around. In any case, the significance of the PIP-box within the RNase H2 complex requires clarification. While RNH201 mRNA expression peaks twice during the yeast cell cycle, during S phase and again during G2/M phase (Arudchandran et al, 2000), the protein accumulates progressively from G1 (where expression is very low) through to M phase (Lockhart et al, 2019). All other, non-catalytic RNase H2 subunit proteins are constitutively expressed throughout the cell cycle, and the complex resides exclusively within the nuclear compartment (Reijns et al, 2012; Lockhart et al, 2019). Thus, RER could theoretically be initiated at any given time in the cell cycle. However, it is conceivable that RER may somehow be temporally regulated, or even coordinated with other DNA repair activities, in order to avoid untimely DNA nick generation and repair synthesis. Although the PIP-box presence in RNase H2 may imply RER coupling to DNA replication, other DNA metabolism and repair processes (such as Okazaki fragment maturation and postreplicative repair) have been found to be postponable until late S/G2 phase without compromising their efficiency (Daigaku et al, 2010; Karras & Jentsch, 2010; Kahli et al, 2019). While RNase H2 did not localize to particular genomic regions in chromatin immunoprecipitations from asynchronously growing yeast cells (Zimmer & Koshland, 2016), it could be cross-linked to telomeres in cells synchronized late in S phase, at times when the bulk of genomic DNA has been replicated (Graf et al, 2017). Consistently, upon fractionation of cell lysates, RNase H2 is more prominently chromatin-associated in G2/M than in S phase, but to a lesser extent in G1 (Lockhart et al, 2019), suggesting that postreplicative chromatin association of RNase H2 may be a more general feature and not just restricted to telomeres. The same work further demonstrated that G2 phase-restricted RNase H2 expression is sufficient to allow its functions both in R-loop removal and RER; while restricting expression to S phase in fact causes defects in R-loop processing as well as RER-related toxicity (Lockhart et al, 2019). It may thus be crucial to limit the peak of RNase H2 activity to a postreplicative period, possibly to minimize generation of DNA double-strand breaks arising from encounters of an oncoming replication fork with RNase H2-induced nicks during S phase. Although risky, RNase H2 activity during S phase does likely still exist and may represent a tolerance pathway for dealing with rNMPs that have not been efficiently removed during the previous cell cycle, and for preventing Top1-mediated genome instability. Follow-up studies to understand the mechanistic basis of RNase H2 cell cycle regulation will be an important next step. Chromatin localization could potentially be regulated by posttranslational modifications of RNase H2 or a cell cycle-regulated RNase H2 inhibitory protein. With respect to the former, a yeast phosphoproteomics screen (Bodenmiller et al, 2010) yielded several phosphopeptides for Rnh202, incidentally the same RNase H2 subunit that also harbors the PIP-box. The identification and characterization of RNase H2 posttranslational modifications could give valuable insights into the regulation of RER activity. In addition, cell cycle-specific chromatin modifications could act as a recruitment signal for RNase H2 and need to be evaluated in this context (Fig 3). Figure 3. Regulation of RER through RNase H2RNase H2 chromatin localization gradually increases throughout S phase but its activity may be kept in check to prevent creation of nicks during replication, where they would be converted into one-ended DSBs by oncoming replication forks. Different regulatory mechanisms (in red) might account for RNase H2 inactivity during S phase. In G2/M, RNase H2 actively processes rNMPs to achieve successful RER. Download figure Download PowerPoint Unrepaired rNMPs affect genome stability Ribonucleotides that permanently reside in the genome can have detrimental consequences on genome stability. Due to the free 2′OH group of the ribose moiety, rNMPs are highly susceptible to spontaneous hydrolysis, thus creating genotoxic single-stranded breaks in the DNA backbone (Li & Breaker, 1999). In yeast, the absence of RNase H2 increases spontaneous mutation rates as well as gene conversion events (Huang et al, 2003; Ii et al, 2011). Moreover, rNMP-dependent gross chromosomal rearrangements (GCRs) are
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