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Wanrooij, P H., Chabes, A. (2019) Ribonucleotides in mitochondrial DNA FEBS Letters, 593(13): 1554-1565 https://doi.org/10.1002/1873-3468.13440

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Permanent link to this version: http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-159088 REVIEW ARTICLE Ribonucleotides in mitochondrial DNA Paulina H. Wanrooij1 and Andrei Chabes1,2

1 Department of Medical and Biophysics, Umea University, Sweden 2 Laboratory for Molecular Infection Medicine Sweden, Umea University, Sweden

Correspondence The incorporation of ribonucleotides (rNMPs) into DNA during genome P. Wanrooij, Department of Medical replication has gained substantial attention in recent years and has been Biochemistry and Biophysics, Umea shown to be a significant source of genomic instability. Studies in yeast and University, 901 87 Umea, Sweden mammals have shown that the two genomes, the nuclear DNA (nDNA) and Tel: +46 707327952 E-mail: [email protected] the mitochondrial DNA (mtDNA), differ with regard to their rNMP content. and This is largely due to differences in rNMP repair – whereas rNMPs are effi- A. Chabes, Department of Medical ciently removed from the nuclear genome, mitochondria lack robust mecha- Biochemistry and Biophysics, Umea nisms for removal of single rNMPs incorporated during DNA replication. In University, 901 87 Umea, Sweden this minireview, we describe the processes that determine the frequency of Tel: +46 736205446 rNMPs in the mitochondrial genome and summarise recent findings regarding E-mail: [email protected] the effect of incorporated rNMPs on mtDNA stability and function. (Received 23 April 2019, revised 9 May 2019, accepted 9 May 2019, available online Keywords: dNTP; genome stability; mitochondrial DNA; ribonucleotides 24 May 2019) doi:10.1002/1873-3468.13440

Edited by Barry Halliwell

DNA rather than RNA is the preferred medium for strand breaks [1]. The presence of isolated rNMPs in long-term storage of genetic information, and this is DNA also alters the local structure and elasticity of partly due to the greater stability of DNA. Together the DNA [2–4], and this can disrupt the recognition with the high fidelity of the replicative DNA poly- and binding by protein factors. On the other hand, merases and the plethora of DNA repair mechanisms transiently-incorporated rNMPs have been suggested that the cell possesses, the chemical and structural sta- to play positive physiological roles, e.g. as markers of bility of double-stranded DNA contributes to the newly-synthesised DNA to guide mismatch repair [5,6] faithful maintenance of our genetic information. How- or to relieve torsional stress created during leading ever, during the past decade the incorporation of strand replication [7]. rNMPs embedded in the genome ribonucleotides (rNMPs) has become recognised as a might thereby influence a variety of fundamental pro- major threat to genome stability. Ribonucleotides dif- cesses including DNA replication, repair, and tran- fer from dNTPs only by the presence of a hydroxyl scription. (OH) group on carbon-2 of their furanose ring. This Our two genomes show striking dissimilarity with seemingly small difference has great implications for regard to ribonucleotide content – while rNMPs are genome stability because the reactive 20-OH group of virtually absent from nDNA, mature mammalian rNMPs can attack the sugar- backbone of mtDNA contains an incorporated rNMP approxi- DNA, and thus incorporation of rNMPs makes the mately every 500–900 depending on the DNA several orders of magnitude more susceptible to source material [8,9]. In vitro, the human mtDNA

Abbreviations AGS, Aicardi-Goutieres syndrome; CSBs, conserved sequence blocks; mtDNA, mitochondrial DNA; nDNA, nuclear DNA; RER, ribonucleotide excision repair; rNMP, ribonucleotide; ssDNA, single-stranded DNA; Top1, topoisomerase 1.

1554 FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies P. H. Wanrooij and A. Chabes Ribonucleotides in mitochondrial DNA polymerase incorporates an rNMP every 2000–2300 Another source of rNMPs in nDNA are the main nucleotides, which is comparable or even lower than nuclear replicative DNA polymerases Pol e and Pol d the rNMP incorporation frequency of nuclear DNA that occasionally incorporate rNMPs during genome polymerases [8–10], although comparisons are compli- replication. Both polymerases exhibit high selectivity cated by variations in assays and reaction conditions. for dNTPs over rNTPs, being at least 100-fold more Rather than differential rNMP incorporation, the dif- likely to insert a dNTP than an rNTP [15,17,18]. This ference in rNMP content between our two genomes is high selectivity for dNTPs is often achieved by use of therefore mainly ascribed to the absence of efficient a bulky steric gate residue that clashes with the 20-OH rNMP removal mechanisms in the mitochondria. group of rNTPs and thus obstructs the entry of the Numerous excellent reviews have summarised the cur- rNTP into the polymerase active site [19,20]. The rent understanding of the roles and consequences of insertion frequency of rNMPs is also influenced by the rNMPs incorporated into the nuclear genome (e.g. ratios of free rNTPs to dNTPs. Free rNTPs are pre- [7,11–13]). Therefore, this mini-review only briefly sent in great excess over dNTPs [15,21], so even the describes general pathways for ribonucleotide incorpo- highly selective replicative polymerases will occasion- ration and removal from nDNA, and then goes on to ally insert rNMPs into their product. Finally, DNA discuss how rNMPs become embedded in mtDNA. polymerases show poor, if any, ability to remove Next, we review the current knowledge on the factors inserted rNMPs with their 30–50 exonuclease activity that determine the frequency and identity of rNMPs in [18,22,23]. Therefore, the majority of inserted rNMPs mtDNA, and we end by considering the implications of will become incorporated into the growing DNA chain the persistence of rNMPs in mature mtDNA. Although as polymerisation continues. The average frequency of our main focus is on mammalian mtDNA, we include rNMP incorporation by Saccharomyces cerevisiae Pol some key findings from the budding yeast system in e in vitro is 1 rNMP per 640–1250 nucleotides inserted, which many landmark discoveries have been made. depending on the assay used. Pol d incorporates fewer rNMPs, with frequencies of 1 rNMP every 720–5000 General pathways for ribonucleotide nucleotides [10,15]. Given that Pol e and Pol d repli- incorporation and removal from cate the vast majority of the eukaryotic genetic mate- nuclear DNA rial, they are estimated to introduce over 10 000 rNMPs per round of replication of the S. cerevisiae nuclear genome [15]. For the mammalian genome, esti- Ribonucleotide incorporation into nDNA mates exceed 3 million rNMPs inserted per round of A major process contributing to the incorporation of replication [15,18,22]. rNMPs in the genome is the priming of DNA replica- Additional processes that might contribute to inser- tion. In the nucleus, the Pol a-primase complex synthe- tion of rNMPs into the genome include DNA repair sises a 20–30 primer that initiates DNA and translesion synthesis. Repair and translesion poly- replication at origins of replication and at each Oka- merases such as Pols l, k, b, s, and f can all use zaki fragment on the lagging strand [14]. The first 7– rNMPs during polymerisation and generally exhibit 10 nucleotides of the replication primer are rNMPs, poorer selectivity for dNTPs over rNTPs than replica- and even the subsequent DNA extension of the primer tive polymerases (reviewed in [7]). Furthermore, the might be interspersed with occasional rNMPs because novel human archaeal-type primase/polymerase Prim- of the relatively high propensity of Pol a to incorpo- Pol that can re-prime stalled replication forks down- rate rNMPs [15]. Given that eukaryotic Okazaki frag- stream of DNA damage or hard-to-replicate sites ments are only about 200 nucleotides in length, prefers to initiate primer synthesis with a ribonu- priming is frequent on the lagging strand, making it cleotide and, although it favours dNTPs during elon- by far the largest source of rNMP incorporation. gation of the primer, can also insert rNMPs when Although replication primers have been thought to be elongating [24,25]. efficiently removed during Okazaki fragment matura- tion, this view has been questioned in light of recent Removal of rNMPs from nDNA findings suggesting that rapid re-association of DNA- binding proteins post-replication might prevent the full Ribonucleotides are removed from the nuclear genome removal of primers synthesised by Pol a-primase [16]. primarily through the action of a dedicated repair Therefore, some rNMPs inserted by Pol a-primase pathway known as ribonucleotide excision repair might escape the Okazaki fragment maturation (RER). Removal is initiated by cleavage on the 50 side machinery and thus remain in mature nDNA. of the rNMP by the endonuclease RNase H2 [26,27].

FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies 1555 Ribonucleotides in mitochondrial DNA P. H. Wanrooij and A. Chabes

Next, strand displacement synthesis by Pol d creates a sites with multiple consecutive rNMPs [32]. Further- displaced rNMP-containing flap that is subsequently more, separation-of-function mutations in mouse removed by Fen1 and/or Dna2, and the repair is com- RNase H2 support the interpretation that the embry- pleted by ligation of the nick by DNA ligase 1 [10]. onic lethality observed in RNase H2 knockouts is RER has high capacity – it removes virtually all caused by single unrepaired rNMPs rather than longer rNMPs present in the nuclear genome of budding RNA/DNA hybrids [38]. yeast even in the presence of dNTP pool imbalances In humans, mutations that lead to partial loss of that result in the increased incorporation of rNMPs RNase H2 activity cause Aicardi-Goutieres syndrome [28]. RNase H2 is critical for initiation of RER at sin- (AGS), a neuro-inflammatory autoimmune condition gle embedded rNMPs, but is additionally involved in that involves activation of the type I interferon system processing longer stretches of rNMPs or R-loops, [39,40]. It appears that while the high levels of unre- which are RNA/DNA hybrids that are the outcome of paired rNMPs found in RNase H2-deficient mice lead an RNA transcript hybridising to DNA [29]. R-loops to an activated DNA damage response and death, the and stretches of four or more consecutive rNMPs are somewhat lower rNMP load observed in AGS mutant also substrates of the RNase H1 enzyme [30], which strains or cell lines instead triggers an innate immune differs from RNase H2 in that it is unable to cleave at response that is dependent on the cGAS/STING path- single rNMPs. way [38,41]. In the absence of RNase H2, numerous rNMPs In contrast to RNase H2, loss of RNase H1 activity remain in the nuclear genome and result in embryonic does not adversely affect nDNA. This likely reflects the lethality in mice and in milder signs of replication fact that the majority of the RNA/DNA hybrid-pro- stress in the yeasts S. cerevisiae and Schizosaccha- cessing activity observed in the cell is actually derived romyces pombe [17,31–33]. The number of rNMPs from RNase H2 [32], whereby loss of RNase H1 can remaining in the nuclear genome when RER is defec- be compensated for by RNase H2 in the nucleus. How- tive has been estimated at 1500 and 1.3 million in ever, RNase H1 is a dual-localised enzyme with two in- S. cerevisiae and mice, respectively [16,32]. In the frame start codons. Translation from the first start absence of RNase H2, some of these rNMPs can be codon produces a mitochondrially-targeted RNase H1, removed by an alternative pathway dependent on while initiation from the second produces the nuclear topoisomerase 1 (Top1) that resolves DNA supercoil- enzyme [42]. Given that RNase H2 is absent in the ing by transiently nicking one DNA strand. Top1 inci- mitochondrial compartment, it cannot compensate for sion at an rNMP creates a 20-30 cyclic phosphate end the loss of mitochondrial RNase H1 activity and hence that must be processed further to allow re-ligation knockout of RNase H1 results in a severe mitochon- [34,35]. When present at repetitive sequences, process- drial phenotype [42,43] that will be discussed in the fol- ing of these ‘dirty’ DNA ends can result in 2–5bp lowing section on mtDNA rNMPs. deletions that in turn trigger checkpoint activation, replication stress, and genome instability [34,36,37]. Ribonucleotides are incorporated The use of this alternative rNMP-removal pathway during mtDNA replication and repair therefore comes at a cost. Features of mtDNA and its replication Defects in rNMP removal have detrimental Mammalian mtDNA is a circular multicopy genome consequences of 16.6 kb that encodes for 13 proteins, 22 tRNAs, As mentioned above, RNase H2 is essential in mam- and 2 rRNAs. Its copy number ranges from a few mals, and knockout of one of its three subunits leads hundred to tens of thousands of mtDNA molecules to embryonic lethality after embryonic day 9. In accor- per cell, depending on the cell type. The 13 polypep- dance with the adverse effects of excess rNMPs on tides encoded by mtDNA are core components of the genome stability, RNase H2-deficient cells exhibit mitochondrial electron transport chain that is essential increased single-stranded DNA (ssDNA) breaks and for efficient energy production. The integrity and suffi- an activated p53-dependent DNA damage response cient copy number of mtDNA is therefore of critical [32,33]. These defects have been attributed to the pres- importance for the cell, as illustrated by the myriad of ence of single or at most double rNMPs rather than disease symptoms associated with mtDNA mutations longer stretches of rNMPs in the genome because or depletion [44,45]. nDNA from RNase H2-deficient MEFs is not sensitive The specifics of mtDNA replication have recently to recombinant prokaryotic RNase HI that digests at been reviewed by others (e.g. [46–48]), and only the

1556 FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies P. H. Wanrooij and A. Chabes Ribonucleotides in mitochondrial DNA aspects that are critical for the discussion of mtDNA illustrated by the susceptibility of purified mtDNA to rNMP incorporation will be summarised here. Mam- cleavage by RNase H2 but not RNase H1 [32]. The malian mtDNA replication relies on a core machinery rNMPs found in mature mtDNA are therefore not consisting of the replicative mtDNA polymerase Pol ɣ, related to the lengthy RNA species that temporarily the helicase TWINKLE, and the mitochondrial coat the displaced H-strand in the RITOLS model nor ssDNA-binding protein mtSSB [49–51]. No dedicated are they longer RNA primers left unremoved after replicative primase has been identified in mammalian replication initiation. The estimated number of rNMPs mitochondria; instead, the replication primers are syn- per double-stranded mtDNA molecule ranges from 36 thesised by the mitochondrial RNA polymerase and 54 rNMPs in cultured HeLa and primary fibrob- POLRMT [52,53]. In addition to these factors, other last cell lines, respectively [9], to 65 rNMPs in mouse proteins are required, e.g. for ligation of the completed liver [8]. In accordance with these numbers, Moss replication products and for relaxing supercoiling [46]. et al. [65] reported a higher frequency of rNMPs in Not only the machinery, but also the mechanism of solid tissues than in cultured cell lines. The location of mtDNA replication differs from that of nDNA. mtDNA rNMPs has been mapped using next-genera- According to the strand-displacement model [54,55], tion sequencing approaches, and they appear to be replication of the two mtDNA strands occurs continu- randomly distributed across the mammalian mitochon- ously on both strands and is asymmetric, with consid- drial genome [9,65]. There is no obvious strand bias in erable delay in replication of the so-called light (L) rNMP frequency between the two strands of mtDNA strand relative to the heavy (H) strand. Replication of isolated from mouse tissues or from cultured human the H-strand initiates in the H-strand origin (OH) cells [8,9]. region and proceeds three quarters of the way around the genome until it exposes the L-strand origin of Priming of mtDNA replication and primer replication (O ). Once in single-stranded form, O L L removal by RNase H1 folds into a stem-loop structure that enables POLRMT to synthesise the L-strand primer that is extended by Conceptually, the rNMPs in mtDNA might derive Pol ɣ [52,56]. The synthesis of both strands then pro- from various processes such as priming, replication, ceeds full-circle, producing two genome-length and/or repair. As previously mentioned, POLRMT mtDNA molecules. synthesises the primer for mtDNA replication. In the

During the first phase of replication before H-strand OH region, transcription from the L-strand promoter synthesis reaches OL, the parental H-strand is dis- that is located a few hundred base pairs upstream of placed as ssDNA. According to the strand-displace- the designated replication origin creates the RNA pri- ment model, the displaced H-strand is coated and mer for replication of the H-strand [53]. In vitro stud- stabilized by mtSSB. This model of mtDNA replica- ies suggest that a prematurely terminated transcript tion has been challenged by the RITOLS model where with a 30 end at CSBII, one of the three conserved the displaced H-strand is instead covered by long sequence blocks (CSBs), forms a G-quadruplex-sta- stretches of transcript RNA [57,58]. However, the long bilised RNA/DNA hybrid with the non-template RNA/DNA hybrids of the RITOLS model are difficult DNA strand [66–69]. This stable RNA/DNA hybrid, to reconcile with the presence of an active RNase H1 termed the mitochondrial R-loop, acts as the replica- in the mitochondria. Furthermore, the in vivo binding tion primer for H-strand synthesis. In support of this pattern of mtSSB corresponds to the pattern expected model of priming, RNA-to-DNA transition sites have according to the strand-displacement model [59], been mapped to the CSB region in vivo [67,70–73]. The 0 whereby the majority of current evidence can be con- majority of the free 5 ends in the OH region do not sidered to favour the strand-displacement model. The contain covalently-attached RNA at their 50 terminus presence of entirely duplex replication intermediates unless RNase H1 is defective, indicating that RNase consistent with conventional coupled leading and lag- H1 is essential for primer removal and that it normally ging strand synthesis has also been reported in mam- functions in an efficient manner [74]. It should also be malian mitochondria [60,61], but the origin or role of noted here that most free 50 ends in this region are these intermediates remains unclear. Further study is located about 100 base pairs downstream of CSBII in therefore required in order to resolve the intricacies of the vicinity of nucleotide position 191, which is for- mtDNA replication. mally defined as OH [67,70,72,73]. The gap between The presence of rNMPs in mature mammalian the observed RNA-to-DNA transitions near CSBII mtDNA was reported as early as the 1970s [62–64]. and the free 50 ends can be explained by further pro- These rNMPs occur singly or at most as pairs, as cessing of the 50 termini of newly-replicated H-strand

FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies 1557 Ribonucleotides in mitochondrial DNA P. H. Wanrooij and A. Chabes molecules by the mitochondrial exonuclease MGME1 to its low processivity and presumably only occasional

[75–77]. A current model for primer removal at the OH involvement in mtDNA synthesis, its contribution can region therefore involves sequential activity of RNase be expected to be minor. Furthermore, rNMP insertion H1, which removes the RNA primer ranging from the by some of the repair or translesion polymerases that L-strand promoter to CSBII, and MGME1, which fur- have been suggested to be present in mitochondria, ther digests the 50 end of the H-strand replication pro- such as Pol b, Pol f, Pol g, and Pol h, cannot be duct from CSBII to OH [75]. excluded until the mitochondrial role of these poly- POLRMT also synthesises the primer at OL. The merases is studied in more detail (reviewed in [82]). structural elements of the OL sequence that are critical for priming have been defined and the process has in vitro Mitochondria lack efficient repair been reconstituted [52,56,78], as has the pro- mechanisms for single embedded cess for removing the RNA primer through RNase H1 rNMPs and Fen1 or a Fen1-like activity [79]. The key role of RNase H1 in primer removal at both mtDNA origins The frequency and identity of mtDNA rNMPs is is highlighted by the fact that RNase H1 knockout partly defined by the dNTP pool mice exhibit progressive mtDNA depletion resulting in embryonic lethality after embryonic day 8.5 [42]. As reviewed above, mtDNA contains single (or at Accordingly, mutations in human RNase H1 cause most double) rNMPs that are broadly scattered over progressive external ophthalmoplegia characterised by both strands (Fig. 1). These rNMPs persist in mtDNA depletion of mtDNA levels and the accumulation of over time and are thus best explained by the absence deletions [43]. Together with the finding that RNA pri- of repair mechanisms rather than by exceptionally fre- mers are retained at both the OH region and OL in quent incorporation during mtDNA replication. RNase H1-deficient cells [74], these observations impli- Indeed, it was found that elimination of RER by dele- cate RNase H1 as the factor responsible for the nor- tion of RNase H2 did not increase mtDNA rNMP fre- mally efficient removal of mtDNA RNA primers. quency in S. cerevisiae [28,83]. Furthermore, analysis of a panel of yeast strains with permanent dNTP pool imbalances revealed that the size and balance of the Incorporation of single rNMPs during mtDNA cellular dNTP pool determines the frequency of indi- replication and repair vidual rNMPs in mtDNA, i.e. an insufficient supply of As with nDNA duplication, single rNMPs are also one dNTP results in increased incorporation of the incorporated during replication of mtDNA by Pol ɣ. corresponding rNMP in mtDNA [28]. In contrast, Biochemical studies indicate that yeast Pol ɣ (Mip1) rNMP levels in nDNA were unaffected by dNTP pool has an rNMP incorporation rate that is comparable to alterations unless RER was eliminated. These results its nuclear counterparts (1 rNMP per 640 nucleotides demonstrate that yeast mitochondria lack efficient in a long-template assay where Pol e and Pol d insert mechanisms for replacing incorporated rNMPs with 1 rNMP per 640 and 720 nucleotides, respectively) dNMPs. Furthermore, given that the base identity of [10,28]. Human Pol ɣ exhibits high discrimination embedded rNMPs was determined by the rNTP/dNTP against rNTPs and inserts only one rNMP per 2300 ratios present in the cell, the majority of mtDNA nucleotides in a similar assay [8,80]. The persistent rNMPs are likely to derive from replication by Pol ɣ rNMPs found in mtDNA are therefore not due to rather than being remnants of replication primers. unusually poor discrimination against rNTPs by the The dNTP pools also affect the type and frequency mtDNA polymerase. As has been found for many of rNMPs embedded in the mtDNA in mammalian other DNA polymerases, the exonuclease activity of cells and tissues. For instance, fibroblasts from Pol ɣ does not contribute to rNMP removal [8,9]. patients with defects in mitochondrial dNTP metabo- Although Pol ɣ is the only replicative DNA poly- lism show altered frequencies of the individual rNMPs merase identified in the mitochondria, there is at least [9]. Furthermore, mice defective in the mitochondrial one other DNA polymerase that is required for inner membrane protein MPV17 that is implicated in mtDNA maintenance under certain conditions. The mitochondrial disease [84–86] exhibit decreased dGTP PrimPol primase-polymerase is partially localised to and dTTP pools in the liver [87] and increased fre- mitochondria [24] and is required for repriming of quency of rGMP embedded in mtDNA [65]. However, mtDNA replication after DNA damage [81]. Repriming the correlation between rNTP/dNTP ratios and and/or translesion synthesis by PrimPol might therefore mtDNA rNMP frequency in mammals might not be contribute to the rNMP load of mtDNA, although due as clear-cut as in budding yeast. For example,

1558 FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies P. H. Wanrooij and A. Chabes Ribonucleotides in mitochondrial DNA

Fig. 1. Differential removal of single rNMPs incorporated during replication of the nuclear and mitochondrial genomes. Replicative polymerases (Pol e: blue; Pol d: green; Pol ɣ: purple) incorporate single rNMPs (red ‘R’) during synthesis of the nascent DNA strand (black line). In the nucleus, RNase H2 (pink oval) initiates ribonucleotide excision repair that removes the rNMPs. In the absence of RNase H2, Top1 (not shown) can cleave at the nDNA rNMPs to aid in their removal. Due to the absence of RNase H2 in the mitochondria, rNMPs are not efficiently removed from the newly-replicated mtDNA. Therefore, even the parental mtDNA strands (grey lines) contain rNMPs. Replication primers are not depicted. although dNTP pools were only found to be altered in rCMP, and rUMP [9,65]. However, the discrimination the liver of MPV17 knockout animals, additional tis- ability of the replicative DNA polymerase also influ- sues (the heart and brain) showed increased mtDNA ences the relative frequency of rNMPs in the mito- rGMP incorporation that could not be explained by chondrial genome. Human Pol ɣ discriminates most an increased rGTP pool [65]. One possible explanation efficiently against rUTP, showing a 77 000-fold prefer- for why rNTP/dNTP ratios might not directly predict ence for dTTP over rUTP, while the discrimination mtDNA rNMP incorporation is that the nucleotide factors for the other rNTPs range from 9300 to 1100 levels measured from cellular or mitochondrial extracts in the order rATP > rCTP > rGTP [80]. When consid- might not accurately reflect the levels available to the ered together, the discriminatory capacity of Pol ɣ and mitochondrial replisome during mtDNA synthesis, for the rNTP/dNTP ratios for each dNTP-rNTP pair example, due to spatial and temporal variations in appear to largely explain the observed relative frequen- dNTP levels. cies of the individual rNMPs in mtDNA. Still, further The dNTP levels fluctuate greatly over the cell cycle, research is called for in order to obtain a complete with the highest concentrations found during S-phase understanding of the factors that determine mtDNA when nDNA replicates [88,89]. However, mtDNA rNMP levels, especially in different cell types and dur- replication is not strictly limited to S-phase or to ing different stages of development. cycling cells [90], and it thus also occurs when cellular dNTP levels are low and rNTP/dNTP ratios are high. The implications of mtDNA rNMPs The rNTP/dNTP ratios from dividing and quiescent mouse Balb/3T3 fibroblasts have been shown to be up As discussed in the first section of this mini-review, the to 10-fold higher in quiescent cells ([21]; Fig. 2). Ele- presence of rNMPs in nDNA has detrimental conse- vated rNTP/dNTP ratios can therefore explain the quences for genome stability [32,33,38]. An additional higher frequency of rNMPs observed in the mtDNA point to consider is that the mitochondrial matrix is of non-dividing vs. cycling cells [65]. Furthermore, the thought to be more basic (pH 8.0) than the nucleus ratios of each dNTP-rNTP pair are a major determi- (pH 7.2) [91], which is expected to render mtDNA nant of the relative frequency of the individual rNMPs more prone to alkaline hydrolysis at sites of rNMP in mtDNA. In line with the high cellular concentration incorporation than nDNA. Given the undisputedly of rATP (a 188-fold and 169-fold excess of rATP over negative effects of persistent rNMPs in nDNA, why dATP in logarithmically growing S. cerevisiae and cul- are rNMPs in mtDNA so well tolerated? One central tured mouse cells, respectively) [15,21], two indepen- reason might be the reverse transcriptase activity of dent next-generation sequencing approaches found Pol ɣ [92,93] that allows it to efficiently replicate DNA rAMP to be the most frequent rNMP in mammalian templates containing incorporated rNMPs. Accord- mtDNA, followed in declining frequency by rGMP, ingly, in vitro analyses have shown that Pol ɣ bypasses

FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies 1559 Ribonucleotides in mitochondrial DNA P. H. Wanrooij and A. Chabes

Cycling cell Quiescent cell Cycling/ rNTP/dNTP quiescent rNTP/dNTP rCTP/dCTP: 28 2.6 rCTP/dCTP: 75 rUTP/dTTP: 45 4.7 rUTP/dTTP: 204 rATP/dATP: 169 6.9 rATP/dATP: 1150 rGTP/dGTP: 85 10.4 rGTP/dGTP: 870

P dNTP rNT dNTP dNTP rNTP

rNTP rNTP rNTP rNTP rNTP rNTP rNTP R rNTP R R R dNTP R R R R rNTP rNTP

R rN R rNTP R TP R R dNTP dNTP R R R R rNTP rNTP P dNTP dNTP rNT dNTP rNTP dNTP

rNTP rNTP rNT rNTP P dNTP

Fig. 2. The rNTP/dNTP ratios in cycling vs. quiescent cells might determine the rNMP frequency of mtDNA. The ratios of each rNTP-dNTP pair have been measured from cycling (left panel) and quiescent (right panel) mouse Balb/3T3 fibroblasts (ratios from [21]). The rNTP/dNTP ratios are 3–10-fold higher in quiescent cells compared to cycling cells (‘Cycling/quiescent’ column), which is expected to result in a higher frequency of rNMPs (red ‘R’s in the bottom panels) in the mtDNA of non-dividing cells. single rNMPs embedded in a DNA template without number. Moss et al. [65] reported the formation of any major negative effects on the processivity or fide- late-onset mtDNA deletions in the brains of MPV17 lity of replication [8,80]. In contrast, multiple consecu- knockout mice that also show increased rGMP incor- tive rNMPs greatly reduce the efficiency of rNMP poration. However, because the overall frequency of bypass by Pol ɣ. However, the polymerase is unlikely mtDNA rNMPs does not appear to be affected (as- to encounter persistent stretches of more than two or sayed from the liver of the same mice), it is at present three rNMPs in vivo because of the presence of RNase unclear whether the deletions are caused by increased H1 in the mitochondria. Additional factors that have rNMP-dependent instability or by some other, yet-un- been suggested to contribute to the tolerance of described mechanism. In summary, the effects of rNMPs in mtDNA include its slower replication rate rNMPs on mtDNA stability and on mtDNA-related [94], its small genome size, and the partial redundancy processes are not yet well-defined. One must also con- conferred by the presence of multiple mtDNA copies sider the possibility that the rNMPs in mtDNA play a per mitochondrion. In support of the latter explana- positive physiological role, as has been suggested for tion, super-resolution microscopy studies have pro- the transient rNMPs in nDNA (discussed above). If vided evidence suggesting that only a subset of the this were to be the case, then a drastic reduction in mtDNA molecules in cultured fibroblasts are undergo- mtDNA rNMPs could have some form of negative ing active replication [95,96], while the other mtDNA consequences. Because even single embedded rNMPs molecules might simply be ‘storage’ of mtDNA. In alter the properties of DNA, they can be envisioned to principle, DNA breaks caused by hydrolysis at rNMPs affect the interaction of DNA-binding proteins with should only be problematic if they occur in the mole- the mtDNA molecule and thus influence processes cule that is actively being replicated. such as mtDNA replication, repair, gene expression, What, if any, is the consequence of an increased packaging, and segregation. mtDNA rNMP frequency? Yeast strains with a decreased mtDNA rNMP load showed improved Conclusions and perspectives mtDNA stability [28]. One possible interpretation of this finding is that rNMPs jeopardise mtDNA stabil- Although the presence of rNMPs in mtDNA has been ity, although alternative explanations also exist given acknowledged for over four decades, we still lack a that the same strains have increased mtDNA copy complete understanding of how these atypical DNA

1560 FEBS Letters 593 (2019) 1554–1565 ª 2019 Federation of European Biochemical Societies P. H. Wanrooij and A. Chabes Ribonucleotides in mitochondrial DNA building blocks influence the stability of the mitochon- 3 Chiu H-C, Koh KD, Evich M, Lesiak AL, Germann drial genome and the many mtDNA-related processes MW, Bongiorno A, Riedo E and Storici F (2014) RNA that are essential for mitochondrial function. With intrusions change DNA elastic properties and structure. increased interest in the role of rNMPs in the nuclear Nanoscale 6, 10009–10017. genome and the introduction of new methods for map- 4 DeRose EF, Perera L, Murray MS, Kunkel TA and ping them, mitochondrial rNMPs have experienced a London RE (2012) Solution structure of the Dickerson boost in interest and have been the subject of several DNA Dodecamer containing a single ribonucleotide. Biochemistry – recent studies. The potential connection of mtDNA 51, 2407 2416. rNMPs to mitochondrial disease via defects in enzymes 5 Ghodgaonkar MM, Lazzaro F, Olivera-Pimentel M, Artola-Boran M, Cejka P, Reijns MA, Jackson AP, involved in mitochondrial nucleotide metabolism Plevani P, Muzi-Falconi M and Jiricny J (2013) makes them even more interesting to study. Ribonucleotides misincorporated into DNA act as Many questions currently remain unanswered. What strand-discrimination signals in eukaryotic mismatch is the consequence of increased rNMPs, and would a repair. Mol Cell 50, 323–332. higher frequency of mtDNA rNMPs be tolerated 6 Lujan SA, Williams JS, Clausen AR, Clark AB and in vivo ? Are rNMP levels really relevant to mitochon- Kunkel TA (2013) Ribonucleotides are signals for drial disease, or are they more of a proxy for mito- mismatch repair of leading-strand replication errors. chondrial dNTP pools? A number of studies have Mol Cell 50, 437–443. demonstrated that increasing dNTP pools elevates 7 Cerritelli SM and Crouch RJ (2016) The balancing act mtDNA copy number [97–99]. While elevated dNTP of ribonucleotides in DNA. Trends Biochem Sci 41, pools might act directly by increasing mtDNA replica- 434–445. tion efficiency and thus mtDNA copy number, it is 8 Forslund JME, Pfeiffer A, Stojkovic G, Wanrooij PH interesting to consider the option that some of the and Wanrooij S (2018) The presence of rNTPs positive effects of increased dNTP pools on mtDNA decreases the speed of mitochondrial DNA replication. copy number might in fact be explained by a PLoS Genet 14, e1007315. decreased frequency of mtDNA rNMPs. The presence 9 Berglund A-K, Navarrete C, Engqvist MKM, Hoberg of fewer rNMPs in the mtDNA should increase its E, Szilagyi Z, Taylor RW, Gustafsson CM, Falkenberg stability and consequently mtDNA copy number. M and Clausen AR (2017) Nucleotide pools dictate the Because the effect of dNTP pools cannot be unlinked identity and frequency of ribonucleotide incorporation from mtDNA rNMP frequency, the individual contri- in mitochondrial DNA. PLoS Genet 13, e1006628. butions of these factors on mtDNA copy number have 10 Sparks JL, Chon H, Cerritelli SM, Kunkel TA, not been determined. Finally, there is a discrepancy Johansson E, Crouch RJ and Burgers PM (2012) Mol between the frequency of rNMPs found in mtDNA RNase H2-initiated ribonucleotide excision repair. Cell 47, 980–986. in vivo (1 rNMP per 500–900 nucleotides) and the fre- 11 Williams JS, Lujan SA and Kunkel TA (2016) quency of rNMP incorporation in vitro (1 rNMP per Processing ribonucleotides incorporated during 2000–2300 nucleotides) [8,9]. Is this discrepancy due to eukaryotic DNA replication. Nat Rev Mol Cell Biol 17, differences in rNTP/dNTP ratios or has the contribu- 350–363. ɣ tion of factors other than Pol been underestimated 12 Vaisman A and Woodgate R (2018) Ribonucleotide in vivo as a source of rNMPs ? We hope for many discrimination by translesion synthesis DNA interesting developments and new insights to address polymerases. Crit Rev Biochem Mol Biol 53, 382–402. these and other pressing questions within the coming 13 Wallace BD and Williams RS (2015) Ribonucleotide years. triggered DNA damage and RNA-DNA damage responses. RNA Biol 11, 1340–1346. Subcell References 14 Pellegrini L (2012) The Pol a-primase complex. Biochem 62, 157–169. 1 Li Y and Breaker RR (1999) Kinetics of RNA 15 Nick McElhinny SA, Watts BE, Kumar D, Watt DL, degradation by specific base catalysis of Lundstrom€ E-B, Burgers PMJ, Johansson E, Chabes A transesterification involving the 2c-hydroxyl group. 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