DNA Replication—A Matter of Fidelity

Molecular Cell

Review DNA Replication—A Matter of Fidelity Rais A. Ganai1,2 and Erik Johansson1,* of Medical Biochemistry and Biophysics, Umea˚ University, SE 901 87 Umea˚, Sweden Hughes Medical Institute, Department of Biochemistry and Molecular Pharmacology, New York University School of Medicine, NY 10016, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2016.05.003 1Department 2Howard

The fidelity of DNA replication is determined by many factors, here simplified as the contribution of the DNA polymerase (nucleotide selectivity and proofreading), mismatch repair, a balanced supply of nucleotides, and the condition of the DNA template (both in terms of sequence context and the presence of DNA lesions). This review discusses the contribution and interplay between these factors to the overall fidelity of DNA replication. Introduction An appropriate level of fidelity during DNA replication ensures the ability of organisms to transfer genetic information from one generation to the next and contributes to the diversity of life. It is essential that the semiconservative duplication of DNA gives a nearly perfect end product, otherwise important genes might carry mutations that lead to disease or cell death. In this review, we will discuss the contributions of the DNA polymerases, mismatch repair proteins, DNA template, and nucleotide pool to the fidelity of DNA replication. DNA replication is a tightly regulated process that begins with the activation of origins of DNA replication (reviewed by RiveraMulia and Gilbert, 2016). Replication forks are established at these origins, and these consist of numerous proteins that contribute to the synthesis of a new chromosome. Numerous proteins make up the Pol III holoenzyme complex in Escherichia coli, and the core of this complex consists of two identical DNA polymerases synthesizing both the leading and lagging DNA strands (McHenry, 2011). The situation is more complex in eukaryotes, and the initial purification of a replication progression complex by Gambus et al. (2006) contained numerous factors, but it was lacking the three eukaryotic replicative DNA polymerases: DNA polymerase a (Pol a), DNA polymerase d (Pol d), and DNA polymerase ε (Pol ε). Recently, a milestone was reached when the minimal set of proteins required for activation of a eukaryotic origin and the establishment of DNA synthesis was identified (Yeeles et al., 2015). Both Pol a and Pol ε were shown to be required, as was expected from a large body of studies over the past 40 years. Pol a consists of four subunits, of which two form the primase and two form the DNA polymerase (Pellegrini, 2012). The primase activity of Pol a is responsible for synthesizing the RNA primer that is required for DNA replication to start, and once this primer is laid down, the enzyme switches to DNA synthesis activity. The roles of Pol d and Pol ε were less clear when the eukaryotic replication machinery was first being investigated, but over the last 10 years, an overwhelming body of evidence has shown that Pol ε copies the leading strand and that Pol d copies the lagging strand during normal DNA replication. Genetic experiments with a Pol ε M644G mutator showed that the Pol ε mutant introduces errors specifically on the leading strand (Pursell et al., 2007), and using the same approach with a Pol d L612M mutant,

it was shown 1 year later that the Pol d mutant introduces errors specifically on the lagging strand (Nick McElhinny et al., 2008). Initially this was only shown with reporter genes in the proximity of specific origins, but a whole-genome study confirmed that the strand bias was true for the Pol d L612M mutant across the entire yeast genome (Larrea et al., 2010). Recently, these studies were challenged by a paper from Prakash and coworkers in which they claimed that Pol d replicates both the leading and the lagging strands and that Pol ε is limited to proofreading errors made by Pol d (Johnson et al., 2015). However, concerns were raised regarding several technical issues in that work, including very low mutation rates, suggesting that the reported results were influenced by suppressor mutations (Burgers et al., 2016). The initial in vivo results suggesting that Pol ε copies the leading strand and Pol d copies the lagging strand during normal DNA replication were also in agreement with biochemical and genetic experiments showing that Pol d facilitates the removal of the RNA primer from each Okazaki fragment when providing Fen-1 (an endonuclease) with an optimal substrate (Garg et al., 2004). The biochemical evidence for this strand bias was further strengthened when a purified CMG complex (the replicative helicase) was shown to selectively position Pol ε on the leading strand (Georgescu et al., 2014). Finally, several independent groups have recently shown that Pol d replicates the lagging strand and Pol ε replicates the leading strand across the genome by mapping the ribonucleotides (rNTPs) that are introduced by Pol d and Pol ε on each strand (Clausen et al., 2015; Daigaku et al., 2015; Koh et al., 2015; Reijns et al., 2015). These examples, and other contributions by many research labs, have shown that there is a strong bias for Pol ε on the leading strand and for Pol d on the lagging strand under normal DNA replication conditions. Thus, Pol d and Pol ε have the greatest impact on the fidelity of DNA replication because together they synthesize at least 90% of the eukaryotic genome (Nick McElhinny et al., 2008). Pol a also has an impact on fidelity because it synthesizes up to about 10% of the genome each time the genome is replicated. Prokaryotes, archaea, and eukaryotes all have mismatch repair systems that recognize errors made by the replicative DNA polymerases. This lowers the mutation rate to a level that is acceptable for the propagation of the species but still allows for slow changes to the genetic code. Molecular Cell 62, June 2, 2016 ª 2016 Elsevier Inc. 745

Molecular Cell

Review

Figure 1. Selection of a Correct Nucleotide The incoming nucleotide is stabilized by interactions in a tight pocket that is formed by the palm domain, fingers domain, template base, and primer base. The three structures are surface representations of Pol a (PDB: 4FYD), Pol d (PDB: 3IAY), and Pol ε (PDB: 4M8O) together with DNA and the incoming nucleotide.

The three major determinants for replication fidelity will first be discussed, and this will be followed by situations in which fidelity is modified by different means. This review will primarily discuss the replicative DNA polymerases under normal DNA replication conditions, but there are also several alternative error-free and error-prone pathways that can come into play when the replication fork is under stress (Lange et al., 2011). Fidelity of DNA Polymerases The fidelity of replicative DNA polymerases is determined by several factors, of which the nucleotide selection step is the major determinant for overall fidelity. DNA polymerases from prokaryotes, archaea, and eukaryotes use the same basic two-metal ion mechanism for nucleotide incorporation into the nascent DNA strand (Johnson, 2010). The DNA polymerase forms a complex with the DNA, and this is followed by the binding of a nucleotide and two metal ions in the polymerase active site of the enzyme (Freudenthal et al., 2012). The binding of a nucleotide within the active site of the polymerase depends on interactions between the nucleotide and the template base, the polymerase active-site residues, the fingers domain of the polymerase, the metal ions, and the 30 -terminal hydroxyl group of the primer strand. These interactions are not fulfilled until after a conformational change occurs within the polymerase, and this is referred to as an induced fit mechanism. If an incorrect nucleotide is bound within the nucleotide binding pocket, the lack of proper geometry within the active site will not support phosphodiester bond formation, and the nucleotide will leave the pocket. This induced fit mechanism of nucleotide selection imparts high selectivity to these polymerases as evidenced by the low rates of single base substitutions and deletions by Pol a, Pol d, and Pol ε (Fortune et al., 2005; Shcherbakova et al., 2003; Zahurancik et al., 2013). The highresolution crystal structures of Pol a, Pol d, and Pol ε in complex with DNA and a dNTP in the polymerase active site show that the incoming nucleotide fits tightly within the active site prior to formation of the phosphodiester bond between the incoming nucleotide and the primer terminus (Figure 1) (Hogg et al., 2014; Perera et al., 2013; Swan et al., 2009). All DNA polymerases have their own unique error signatures, 746 Molecular Cell 62, June 2, 2016

which emphasizes the different influences that these interactions have in determining fidelity. The in vitro nucleotide selection fidelities of Pol a, Pol d, and Pol ε have been estimated to be about 1 error per 104–105 incorporated nucleotides (Fortune et al., 2005; Kunkel et al., 1991; Shcherbakova et al., 2003). Recent in vivo estimates, however, suggest that the base selectivity is actually many orders of magnitude higher (St Charles et al., 2015). On average, it was determined that the wrong base is incorporated into the yeast genome only once per 107 nucleotides. This means that in yeast only one misincorporated base appears on average each time the DNA is duplicated. In addition, proofreading and mismatch repair increase the fidelity under unstressed conditions to a level where only one replication error occurs for every 250 cell generations (Lujan et al., 2014). The remarkably high level of fidelity during nucleotide selection, and the discrepancy between the in vitro and in vivo estimates, can be explained in several ways. The in vitro error rates could be overestimated due to suboptimal salt concentrations or pH. Another possibility is that some specific protein interactions that influence fidelity are missing in vitro. An enticing possibility is that there could be yet undiscovered error repair mechanisms that are as important as proofreading or mismatch repair. Proofreading Proofreading by DNA polymerases is the second mechanism that ensures high fidelity during DNA replication, and this can be divided into two steps. The first step is to recognize a misincorporated nucleotide, and the second step is to remove it with the help of the intrinsic 30 –50 exonuclease activity that is present in the replicative DNA polymerases. The fidelity of replicative DNA polymerases is determined by the fine balance between the ability to extend the nascent strand and the removal of incorrectly added nucleotides. A correct nucleotide favors continued DNA synthesis in the polymerase site, and an incorrect nucleotide stalls DNA synthesis and the nascent strand is transferred to the exonuclease site. For an in-depth review on this topic, please see Reha-Krantz (2010). The ability to extend a mismatched primer terminus also determines the fidelity of the DNA polymerase. If it is unable to extend the mismatch, the

Molecular Cell

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Figure 2. Possible Outcomes after Pol d or Pol ε Make a Replication Error

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replicative DNA polymerase

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DNA replication during next S-phase

(A) The replicative DNA polymerase inserts a wrong nucleotide or creates a 1 nt insertion or deletion. (B) The replicative DNA polymerase dissociates as a consequence of stalling. This allows another protein with exonuclease activity to remove the replication error, which is followed by continued synthesis by the replicative DNA polymerase. (C) Stalling allows the replicative DNA polymerase to transfer the 30 end of the primer to the intrinsic exonuclease site. The 30 end returns to the polymerase site after the replication error is removed, and DNA replication continues. (D) The replicative DNA polymerase inserts a wrong nucleotide and adds additional nucleotides. (E and F) This leaves a replication error that must be corrected by mismatch repair (E) or else a permanent change in the DNA sequence will be established the next time the genome is duplicated (F).

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DNA polymerase will either transfer the 30 end to the intrinsic exonuclease site or dissociate and expose the 30 end to other proteins that can correct the error (Figures 2A–2C). Evidence for such a mechanism comes from the observation that errors made by Pol a and Pol ε can be corrected by Pol d (Flood et al., 2015; Pavlov et al., 2006). In contrast to proteins such as transcription factors, DNA polymerases must interact with the DNA in a sequence-independent manner. The major points of contacts are along the phosphate backbone of the DNA and through a hydrogen bond network with structurally important water molecules in the minor groove of the duplex DNA (Figures 3A–3C). Not much is known regarding the contribution of the interactions with the phosphate backbone, but studies on bacteriophage RB69 DNA polymerase have shown that the hydrogen bond network along the minor groove is important for the fidelity of the enzyme (Xia et al., 2012). The thumb domains in B-family polymerases contain a KKRY motif that has been shown to be important for stabilizing the DNA within the polymerase active site (Blasco et al., 1995), and high-resolution structures of B-family polymerases have shown that one of the lysines in that motif contacts the primer base at the n–2 position upstream from the most recently formed base pair (Figure 3D) (Hogg et al., 2014; Perera et al., 2013; Swan et al., 2009; Wang et al., 1997). Loss of this interaction—as might occur when an incorrect nucleotide has been incorporated onto the end of the primer—destabilizes the 30 end of the primer in the polymerase active site and results in melting of the primer end and a switch to the exonuclease active site (Ganai et al., 2015; Zakharova et al., 2004). Pol d and Pol ε also have a second point of interaction between an arginine residue in the thumb domain and the bases at position n–4/n–5 (Figure 3D) (Hogg et al., 2014; Swan et al., 2009). The loss of this interaction results in a switch of the primer end to the exonuclease site in Pol ε (Ganai et al., 2015). These sequence-independent contacts between the DNA polymerase and the DNA allow the enzyme to determine whether the nascent strand is correctly paired with the template

or not. Interestingly, A-family DNA polymerases might sense mismatches by long-range distortions in the DNA over the first four base pairs (Johnson and Beese, 2004). Whether B-family DNA polymerases are also influenced by similar long-range distortions in the DNA is unclear. If an error has occurred, a large conformational change takes place within the B-family polymerase to allow 4 or 5 base pairs to melt and for the 30 end of the nascent strand to be transferred about 40 A˚ from the polymerase active site to the exonuclease site (Franklin et al., 2001; Shamoo et al., 1995). After the removal of one or several nucleotides from the primer end, the 30 end is transferred back to the polymerase site. Amino acid substitutions that affect this switch also affect the fidelity of the DNA polymerases because aberrant switching can either increase or decrease the fidelity of the enzyme (Reha-Krantz, 2010). Mismatch Repair Errors that evade the replicative DNA polymerases’ proofreading functions are corrected by a separate system called mismatch repair (Figures 2D–2F) (Kunkel and Erie, 2015). Mismatch repair involves specific proteins that recognize various types of replication errors that can be divided into mismatches, insertions, and deletions. After detection of the replication error, the mismatch repair system must determine which strand should be corrected, which is referred to as strand discrimination. The template strand should be left intact, and a nick must be created on the nascent strand that contains the replication error. In E. coli, it has been shown that a methyl group on the template strand directs MutH to make an incision on the opposite strand from the misincorporated nucleotide, and this allows an exonuclease to remove the portion of the newly replicated DNA that includes the replication error. After that, a DNA polymerase fills the gap and a DNA ligase seals the final nick (Kunkel and Erie, 2015). In eukaryotes, replication errors are recognized by MutSa and MutSb (Kunkel and Erie, 2015). The strand discrimination signal was unknown for many years, but now there are three processes Molecular Cell 62, June 2, 2016 747

Molecular Cell

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Figure 3. Interaction between DNA and DNA Polymerase (A–C) Surface representation of all amino acids located within 4 A˚ of the DNA in structures with (A) Pol a (PDB: 4FYD), (B) Pol d (PDB: 3IAY), and (C) Pol ε (PDB: 4M8O). The majority of the interactions are with the sugar-phosphate backbone. (D) K967 in Pol ε (green) and K814 in Pol d (blue) contact the base at position n–2 via the minor groove of the DNA. R988 in Pol ε (green) and R839 in Pol d (blue) contact bases at position n–4/n–5 via the minor groove of the DNA. Duplex DNA from both structures is shown in orange.

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that have been suggested to perform such a role. First, it was discovered that MutLa has a PCNA-dependent endonuclease activity that allows strand-specific cleavage (Kadyrov et al., 2006). MutLa cleaves the strand with the replication error on either side of the mismatch in a MutSa–, RFC-, and PCNAdependent manner. Genetic experiments in yeast demonstrated that inactivation of MutLa endonuclease activity results in an almost complete loss of mismatch repair activity (Kadyrov et al., 2007). Second, nicks created during ribonucleotide excision repair were recently suggested to provide an alternative strand discrimination signal for mismatch repair on the leading strand, but the inactivation of RNaseH2 had a much smaller effect on mismatch repair than was expected, suggesting that such nicks contribute to a lesser extent than the nicks created by MutLa (Ghodgaonkar et al., 2013; Lujan et al., 2013). Third, nicks that separate Okazaki fragments on the lagging strand have also been suggested to act as a strand discrimination signal for mismatch repair (Pavlov et al., 2003). In addition to the possible strand-discrimination signals, there are three models describing how the replication error is removed from the nicked strand depending on whether the nick is located 50 or 30 of the lesion. In the first model, exonuclease 1 (Exo1) removes the strand with the replication error in a 50 –30 direction (Kadyrova and Kadyrov, 2016). In the second model, it has been suggested that strand displacement synthesis by Pol d and Pol ε together 748 Molecular Cell 62, June 2, 2016

with flap cleavage can contribute to removal of the mismatch in the 50 –30 direction (Kadyrov et al., 2009). A third model involves exonuclease-dependent degradation of DNA by Pol ε or Pol d in the 30 –50 direction. This third model is supported by the observation that a combination of Exo1 deletion and exonuclease-deficient polymerases results in almost complete loss of mismatch repair (Tran et al., 1999). In the final steps of mismatch repair, a DNA polymerase fills the gap and DNA ligase seals the nick. Genetic and biochemical studies have implicated both Pol ε and Pol d in eukaryotic mismatch repair, although the role of Pol ε is not as well understood (Longley et al., 1997; Tran et al., 1999). Mismatch repair corrects errors with different efficiency at different positions in the genome. Errors in the lagging strand are more efficiently repaired by mismatch repair compared to those in the leading strand, and it has been proposed that the high density of 50 ends at Okazaki fragments and the higher density of PCNA on the lagging strand might stimulate mismatch repair activity (Pavlov et al., 2003; St Charles et al., 2015). Deep sequencing of yeast strains also reveals a great variation in the efficiency with which mismatch repair corrects specific mismatches. For example, mismatch repair was shown to provide a 490-fold correction factor for template C-T mismatches, but only a 6-fold correction factor for template T-T mismatches (St Charles et al., 2015). The sequence context in which a specific mismatch is positioned also affects the efficiency with which mismatch repair corrects replication errors. In one example it was shown that a T-T mismatch, located adjacent to an AT-rich triplet repeat, was inefficiently corrected by mismatch repair. Changing the sequence of the adjacent AT-rich triplet repeat restored mismatch repair correction of the T-T mismatch to >95% efficiency (Lujan et al., 2012). The complexity of the contribution from nucleotide selection, proofreading, and mismatch repair to the overall fidelity becomes quite apparent when whole-genome sequencing is used in detailed studies of replication fidelity. Earlier in vitro studies showed that Pol ε has higher fidelity than Pol d (Fortune et al., 2005). The current in vivo studies show in general that the higher fidelity of Pol ε compensates for less efficient mismatch repair on the leading strand and that the slightly lower fidelity of Pol d is compensated for by more efficient mismatch repair

Molecular Cell

Review Figure 4. Consequences of Ribonucleotide Incorporation in the Genome (A) Ribonucleotide excision repair (RER) removes the incorporated ribonucleotides and involves at a minimum RNase H2, RFC, PCNA, Pol d/ Pol ε, Fen-1, Exo1, and DNA ligase. (B) Aborted ligation by DNA ligase adds AMP to the 50 of the ribonucleotide. This can either occur during RER or the Okazaki fragment maturation process. Aprataxin removes the AMP, and the resulting DNA substrate can enter the RER pathway. (C) Top1 creates unligatable ends on the DNA, and this leads to 2- to 5-nt deletions.

A

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on the lagging strand (St Charles et al., 2015). Thus, both strands have approximately the same overall fidelity, but the efficiency by which errors are corrected at specific positions in the genome varies dramatically. The complex relationships between the replicative DNA polymerases, mismatch repair proteins, and exonucleases—and how these influence replication fidelity— make this a demanding field of study, but the insights that can be gained from deep sequencing has the potential to provide not only important information about the fidelity of DNA replication but also how the replication fork responds to challenges during DNA replication. Modifiers of Fidelity Ribonucleotides The concentration of rNTPs is much higher than dNTPs in vivo. Although it is difficult to determine the precise concentrations, the relative amount of each rNTP was found to be about 36- to 190-fold higher than its corresponding dNTP (Nick McElhinny et al., 2010b; Sabouri et al., 2008). Thus, it is a major challenge for DNA polymerases to discriminate between rNTPs and dNTPs. Family B DNA polymerases contain a tyrosine that forms a steric gate that excludes rNTPs from the active site (Joyce, 1997). Despite having this steric gate, it was recently found that the large excess of rNTPs over dNTPs still results in incorporation of rNTPs into DNA by Pol a, Pol d, and Pol ε (Nick McElhinny et al., 2010a, 2010b). High concentrations of rNTPs have also been shown to decrease the rate of DNA synthesis, which further emphasizes that rNTPs are a challenge for the DNA polymerases (Yao et al., 2013). Estimates show that Pol ε incorporates one rNTP for every 1,250 dNTPs, whereas Pol d and Pol a incorporate an rNTP for every 5,000 and 625 dNTPs, respectively (Nick McElhinny et al., 2010a, 2010b). Thus, rNTPs are the most common form of DNA lesion inside the cell surpassing all other types of DNA lesions, in part because DNA polymerases have been shown to proofread

rNTPs very inefficiently (Clausen et al., 2013b; Williams et al., 2012). Several pathways are activated as a consequence of rNTPs in the genome, and these are mediated by RNase H2, topoisomerase I, and post-replication C repair mechanisms (Figures 4A–4C) (Kim et al., 2011; Lazzaro et al., 2012; Nick McElhinny et al., 2010a; Williams et al., 2013a). RNase H2-mediated ribonucleotide excision repair has been successfully reconstituted in vitro with purified proteins (Figure 4A) (Sparks et al., 2012). First, RNase H2 makes a strand incision on the 50 end of the incorporated rNMP, and this is followed by extension from the 30 -OH by Pol d and subsequent displacement of the rNMP in a PCNA-dependent manner. The resulting flap is cleaved by Fen1, and nicks are sealed by DNA ligase I. Exo1 and Pol ε were also shown to be able to participate in ribonucleotide excision repair, although less efficiently than Fen1 and Pol d. Deletion of the RNH201 gene (catalytic subunit of RNase H2) but leads to an increased mutation rate due to an error-prone pathway on the leading strand (Williams et al., 2015a). rNTPs in the DNA that evade the ribonucleotide repair pathway can be cleaved by Topoisomerase 1, which results in nicks with unligatable ends (Figure 4C) (Williams et al., 2013a). A consequence of this is checkpoint activation, slow progression through the cell cycle, sensitivity to hydroxyurea, and elevated rates of 2- to 5-nt deletions (Clark et al., 2011; Kim et al., 2011; Nick McElhinny et al., 2010a). Nicks with a 50 -ribose cannot be accurately sealed by DNA ligase, which instead generates chemically adducted, abortive toxic adenylated 50 end DNA lesions (50 -adenylated ribose [50 -AMP]) (Figure 4B). Aprataxin binds to these structure and removes the adduct to create a nick that can be closed by DNA ligase (Tumbale et al., 2014). Another consequence of unrepaired rNTPs in the template strand is that Pol a, Pol d, and Pol ε have difficulties bypassing these lesions (Clausen et al., 2013a; Watt et al., 2011). Instead, cells depend on post-replication repair and Pol z to complete DNA replication (Lazzaro et al., 2012). Several biological consequences of having rNTPs in the genome have been reported in the literature. Mouse embryonic fibroblasts lacking RNase H2 have increased genomic instability (Reijns et al., 2012), and knockout of Rnaseh2b or Rnaseh2c (encode subunits of RNase H2) in mice leads to embryonic lethality (Hiller et al., 2012; Reijns et al., 2012). In humans, Molecular Cell 62, June 2, 2016 749

Molecular Cell

Review mutations in three genes encoding the subunits of RNase H2 are associated with a neuroinflammatory disorder called AicardiGoutie`res syndrome (Crow et al., 2006), and the neurological disorder Ataxia with Oculomotor Apraxia 1 is associated with the loss of aprataxin that resolves the RNA-DNA structures (Moreira et al., 2001). Interestingly, incorporated rNTPs appear to direct MMR proteins to the nascent strand during leading strand synthesis (Ghodgaonkar et al., 2013; Lujan et al., 2013). This was shown in in vitro studies using human cell extracts that revealed that a mismatch and the rNTP need to be within 1 kb from each other in order for mismatch repair proteins to track along the DNA for removing mismatches (Ghodgaonkar et al., 2013). Genetic studies by Kunkel and colleagues corroborate the idea that RNase H2-dependent removal of rNTPs on the leading strand might signal the mismatch repair system to correct mismatches within the vicinity of the rNTP (Lujan et al., 2013). Similar observations have been made in Bacillus subtilis where incorporated rNTPs can contribute to a strand discriminatory signal for mismatch repair (Yao et al., 2013). In Schizosaccharomyces pombe, two rNTPs are incorporated into the lagging strand close to the mat1 locus, and they remain unrepaired throughout the cell cycle (Vengrova and Dalgaard, 2004). During the next round of DNA replication, the presence of the two consecutive rNTPs in the template strand induces a structural change in the DNA helix that stalls the leading-strand polymerase, Pol ε, and this initiates mating-type switching. Structural studies on bacteriophage RB69 gp43 DNA polymerase showed that the presence of two consecutive rNTPs with the ribose in the endo-conformation causes the DNA polymerase to stall at position n–2 (Clausen et al., 2013a). Deoxyribonucleotides The concentration of each dNTP in the cell influences the selection of the correct nucleotide by DNA polymerases. Both a general increase in dNTP concentration with a balanced supply of the four dNTPs and an unbalanced supply of dNTPs have been shown to affect DNA replication fidelity (Buckland et al., 2014; Kumar et al., 2011; Mertz et al., 2015; Williams et al., 2015b). Analogous to the concentration effect of rNTPs on fidelity, an elevated concentration of a specific dNTP will increase the chance that it will be misincorporated despite the fact that not all of the interactions in the active site are fulfilled. Second, an increased concentration of dNTPs drives the extension of mismatches at the cost of proofreading by tipping the balance away from proofreading (Reha-Krantz, 2010). Detailed studies of yeast strains with modified dNTP pools have just begun to unveil how the concentrations of dNTPs are adapted to achieve an appropriate level of fidelity during DNA replication. Ribonucleotide reductase catalyzes the rate-limiting step in the production of all four dNTPs (Reichard, 1988), and both the overall concentration and the relative concentrations in wildtype eukaryotic cells (dT > dA = dC > dG) are maintained primarily by ribonucleotide reductase activity. Strains with engineered ribonucleotide reductase mutants that result in defined differences in relative dNTP concentrations accumulate mutations within different sequence contexts (Kumar et al., 2011; Watt et al., 2015). Interestingly, it appears that the replication errors are similar regardless of whether they are made by Pol d or Pol ε. This is interesting because it was previously shown that 750 Molecular Cell 62, June 2, 2016

Pol d and Pol ε have distinct error signatures (Fortune et al., 2005; Shcherbakova et al., 2003). Thus, the induced replication errors must occur by a general mechanism that is independent of the selection for the correct nucleotide. It was proposed that the mechanism for incorporation of mismatches is driven by the unbalanced pools. For example, in a sequence context where a mismatch is followed by G or A in the template, an increased concentration of dCTP and dTTP will drive the reaction forward through the incorporation of dCMP or dTMP (Watt et al., 2015). Thus, the next nucleotide effect will be stronger than the unique active site properties of the specific DNA polymerase. There is also a very strong correlation between the rate of single-nucleotide deletions and the length of a homonucleotide run (Lujan et al., 2015). Unbalanced dNTP pools enhance this correlation through the influence of the nucleotide that comes immediately after the homonucleotide run (Kumar et al., 2011; Watt et al., 2015). Thus, carefully regulated dNTP concentrations are critical for normal fidelity during DNA synthesis. A recent publication demonstrated that some colon cancers have inactivating mutations in SAMHD1, a protein responsible for the degradation of dNTPs, and these tumors are expected to have increased dNTP levels and elevated mutation rates (Rentoft et al., 2016). It has yet to be determined, however, if alterations of the dNTP pools can directly elevate the risk of developing cancer. Unrepaired DNA Lesions in the Template DNA lesions that are left unrepaired in the template strand are a serious threat to the stability of the genome. It lies outside the scope of this review to discuss all types of DNA lesions that cause challenges for the replicative DNA polymerases, and interested readers can find more detailed information in many excellent reviews (e.g., Lange et al., 2011; Xu et al., 2015). A classic example of a DNA lesion that affects replication fidelity is the hydrolysis of cytosine into uracil. DNA polymerases will faithfully incorporate dATP opposite the uracil, and this results in a permanent change from C to T. Another example is oxidation of G that gives 8-oxo-G, across which DNA polymerases tend to favor incorporation of dATP instead of the correct dCTP. In this case the replication error will result in a permanent change from G to T. Analogous to the general variation in errors produced during nucleotide selection, misreading efficiency also varies for 8-oxo-G between DNA polymerases. There are reported ratios of dA:dC insertion ranging from 200:1 to 1:7 for different DNA polymerases (references in McCulloch et al., 2009), and both Pol ε and Pol d preferentially insert dATP over dCTP opposite 8-oxoG (McCulloch et al., 2009; Sabouri et al., 2008). It was recently suggested that DNA polymerases are able to insert 8-oxo-dGTP with a sufficiently high frequency to cause lethality in cancer cells when MTH1 is inhibited (Gad et al., 2014). MTH1 removes oxidized dNTPs such as 8-oxo-dGTP from the dNTP pool, and inhibition of MTH1 leads to increased concentrations of 8-oxo-dGTP. It was shown that mammalian Pol d inserts 8-oxo-dGTP with a three orders of magnitude lower efficiency than dGTP during steady state kinetics, and it was proposed that only one 8-oxo-dGTP would be inserted each time the mammalian genome is duplicated (Einolf and Guengerich, 2001). It has not yet been shown with what efficiency the

Molecular Cell

Review

Figure 5. Cancer-Associated Amino Acid Substitutions in Pol ε The majority of the cancer-associated amino acid substitutions in the exonuclease domain of Pol ε are found in central hydrophobic regions, the cavity where single-stranded DNA binds, and within the catalytic site. Cancerassociated amino acids are highlighted in red, with the given position of the most frequently reported P286 residue based on an alignment with yeast Pol ε. The single-stranded DNA from the structure of the exonuclease domain of T4 bacteriophage (PDB: 1NOY) (Wang et al., 1996) is modeled into the exonuclease domain in yeast Pol ε (PDB: 4M8O).

replicative DNA polymerases incorporate 8-oxo-dGTP in the presence of dGTP. DNA repair enzymes such as Pol b have been shown to insert 8-oxo-dGTP in the absence of dGTP (Freudenthal et al., 2015). However, concerns have recently been raised by two independent studies that were unable to reproduce the initial report that cancer cells are sensitive to inhibition of MTH1 activity (Kettle et al., 2016; Petrocchi et al., 2016). DNA lesions in the template not only affect the selection of the incoming nucleotide, they can also activate a checkpoint that leads to elevated dNTP concentrations. Elevated dNTP concentrations improve survival in yeast, increase the fork progression rate, and stimulate DNA polymerases to bypass DNA lesions in the template (Chabes et al., 2003; Poli et al., 2012; Sabouri et al., 2008). In some cases, error-prone DNA polymerases are engaged to bypass the DNA lesions, but replicative DNA polymerases also have a higher propensity to bypass DNA lesions in the presence of increased dNTP pools (Sabouri et al., 2008). As discussed above, this increased survival comes at the cost of a higher mutation rate. Genomic Instability and Cancer Genomic instability is one of the hallmarks of cancer. Thus, it has long been discussed whether cancer cells, in general, carry genetic changes that provide a higher mutation rate that fuels the heterogeneity within tumors (Fox et al., 2013). Two decades have passed since it was established that mutations that inactivate mismatch repair genes do, in fact, lead to a predisposition for hereditary nonpolyposis colorectal cancer. Loss of mismatch repair is also common in sporadic colon cancers and results in microsatellite instability and high levels of mutations in the genome. It was shown early on in yeast that proofreading by

Pol ε and Pol d is as important as mismatch repair for the mutation load (Morrison and Sugino, 1994). Evidence that the exonuclease activity of Pol ε and Pol d protects mammals from cancer was first demonstrated in mice. Homozygotic mice with exonuclease-deficient Pol d died within 8 months either due to thymic lymphoma, skin tumors, lung adenocarcinomas, or teratomas. Mice carrying the homozygous mutations in the exonuclease domain of Pol ε predominantly developed gastrointestinal tumors and lymphoma (Albertson et al., 2009; Goldsby et al., 2002). These studies in mice also suggested that point mutations could drive cancer development in addition to the large increase in insertions and deletions found in mismatch repair-deficient tumors (Preston et al., 2010). The first evidence that Pol ε is associated with human cancer came when The Cancer Genome Atlas exome sequenced 224 solid colorectal cancer tumors and an independent group analyzed the exomes of 72 colorectal cancers (2012; Seshagiri et al., 2012). A subgroup of the tumors were ultramutated, and mutations in mismatch repair genes and the gene encoding the catalytic subunit of Pol ε, POLE, were overrepresented. In contrast to the mismatch repair-deficient tumors, tumors with mutations in the exonuclease domain of POLE did not show microsatellite instability. The role of proofreading was further strengthened when it was found that mutations in the exonuclease domains of POLE and POLD1 (the gene encoding the catalytic subunit of Pol d) gave a predisposition for hereditary colon cancer (Palles et al., 2013). A picture has now emerged from numerous studies that POLE mutations are more frequently observed than POLD1 mutations in sporadic and hereditary cancers. Studies from sporadic cancers show that POLE exonuclease domain mutations are found in 1%–2% of sporadic colon cancers and 7%–12% of sporadic endometrial cancers (Rayner et al., 2016). They are also found in ultramutated tumors of the stomach, breast, pancreas, ovary, brain, and uterine carcinosarcomas. Most of the described mutations in the POLE exonuclease domain lead to amino acid substitutions in or adjacent to the region where single-stranded DNA is predicted to bind or in the exonuclease catalytic site (Figure 5). The most common single substitution P286R/H is located in a loop that is not in the immediate vicinity of the single-stranded DNA, but appears to form a lid on top of the exonuclease domain (Hogg et al., 2014; Kane and Shcherbakova, 2014). This mutated proline is highly conserved and is also present in yeast Pol ε. This mutant became very interesting when it was shown to result in a polymerase with a much higher mutation rate than yeast Pol ε with a completely dead exonuclease domain (Kane and Shcherbakova, 2014). The mechanism behind this exceptionally high mutation rate is still unclear, and it will be very interesting to see if this particular mutant can provide new knowledge about how the replication fork functions. As discussed above, the dNTP concentrations influence the fidelity of DNA polymerases, and this is also true for yeast cells carrying mutations in Pol d and Pol ε. Decreased dNTP concentrations suppress the mutator phenotype of an exonucleasedeficient Pol ε allele in yeast (Williams et al., 2015b), and a colon cancer-associated mutation in Pol d, POLD1-R688W, was shown to replicate DNA with decreased fidelity (Daee et al., 2010). In addition, it was recently shown that the corresponding Molecular Cell 62, June 2, 2016 751

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Review tation rate will result in a sufficient number of changes in cellular proteins that the immune system will recognize the cancer cells as non-self and kill them (Bellone et al., 2015). nucleotides

nucleotide selection

proofreading

1 error / 109-10 nts

template

mismatch repair

Figure 6. Determinants of DNA Replication Fidelity There is a balance between nucleotide selectivity, proofreading efficiency, mismatch repair, nucleotide pools, and the effect of the nature of the DNA template (sequence context and the presence DNA lesions) that determines the overall fidelity of DNA replication. Amino acid substitutions in the DNA polymerase might alter the nucleotide selectivity and proofreading to either increase or decrease the fidelity to give an antimutator or mutator phenotype, respectively. Inactivation of mismatch repair gives decreased fidelity. The sequence context and the presence of DNA lesions in the DNA template will have an effect on replication fidelity. Altered dNTP pools can result in both increased and decreased fidelity and can have an impact on all DNA synthesis that occurs in the cell.

yeast mutant, pol3-R696W, causes checkpoint activation that leads to higher dNTP pools (Mertz et al., 2015). The increased dNTP concentration enhances the inherent infidelity of the pol3-R696W mutant, and this suggests that secondary effects from replication defects can create vicious cycles that drive genomic instability. It is not only altered nucleotide pools that can enhance genomic instability, and defects in DNA repair systems, lesion bypass, and expression levels of error-prone DNA polymerases have also been suggested to play a role (Lange et al., 2011). The increased mutation rates that are caused by decreased fidelity during DNA replication have two major consequences. First, the risk for acquiring mutations that change the normal cell into a cancer cell increase, and the time that it takes for this to occur is shortened. This can be seen in Lynch syndrome patients with nonfunctional mismatch repair who have a high incidence of colon cancer before age 40. Second, patients with ultramutated tumors in some cases have a better prognosis than patients carrying tumors with a normal mutation load (Cancer Genome Atlas Research et al., 2013). One example of this is a subgroup of endometrial cancers with ultramutated genomes and an exonuclease domain mutation in POLE. The reason why patients with ultramutated tumors have a better prognosis is still not clear. However, in general the fitness of a cell will decrease when too many mutations have been acquired. This is exemplified in yeast where mutation rates can increase to a level where a further increase causes cell death, which is referred to as the error extinction rate (Williams et al., 2013b). In the case of endometrial cancers, it has been suggested that the high mu752 Molecular Cell 62, June 2, 2016

Outlook The fidelity of DNA replication is determined by many factors, here simplified as the contribution of the DNA polymerase (nucleotide selectivity and proofreading), mismatch repair, a balanced supply of nucleotides, and the condition of the DNA template (both in terms of sequence context and the presence of DNA lesions) (Figure 6). It was proposed some 40 years ago that cancer cells generate mutations at a higher rate than normal cells (Loeb et al., 1974), and this has now become accepted as a model for cancers that are mismatch repair deficient or proofreading deficient (Pol ε and Pol d exonuclease domain mutants). However, it is still not clear whether the cancer mutator hypothesis can be generalized to all cancers. This is due in part to the complex interaction between factors that influence the fidelity of DNA replication. Over the course of evolution, a balance has been established between nucleotide selectivity, proofreading, mismatch repair, nucleotide concentrations, and difficult-toreplicate DNA templates (e.g., certain sequence contexts or the presence of DNA lesions) that together allow for sufficiently accurate duplication of the genome. The fidelity of replication is not identical across the genome, and the different factors contribute to varying degrees of fidelity at different locations in the genome. Thus, even slight changes in this balance might result in decreased fidelity. With next-generation sequencing techniques, studies of DNA replication fidelity have moved into whole-genome studies that might provide an increased understanding of the mechanisms through which DNA replication is carried out. Furthermore, the insights that will be made might also clarify whether all cancers have a higher mutation rate and whether there is an optimal mutation rate for cancer cells that does not negatively affect fitness but still promotes the evolution of aggressive cancer cells and drug resistance (Schmitt et al., 2015). ACKNOWLEDGMENTS We thank Vimal Parkash for help with the production of Figures 1 and 5. This work was supported by Cancerfonden, the Swedish Research Council, and the Knut and Alice Wallenberg Foundation (to E.J.). REFERENCES Albertson, T.M., Ogawa, M., Bugni, J.M., Hays, L.E., Chen, Y., Wang, Y., Treuting, P.M., Heddle, J.A., Goldsby, R.E., and Preston, B.D. (2009). DNA polymerase epsilon and delta proofreading suppress discrete mutator and cancer phenotypes in mice. Proc. Natl. Acad. Sci. USA 106, 17101–17104. Bellone, S., Centritto, F., Black, J., Schwab, C., English, D., Cocco, E., Lopez, S., Bonazzoli, E., Predolini, F., Ferrari, F., et al. (2015). Polymerase ε (POLE) ultra-mutated tumors induce robust tumor-specific CD4+ T cell responses in endometrial cancer patients. Gynecol. Oncol. 138, 11–17. Blasco, M.A., Me´ndez, J., La´zaro, J.M., Blanco, L., and Salas, M. (1995). Primer terminus stabilization at the phi 29 DNA polymerase active site. Mutational analysis of conserved motif KXY. J. Biol. Chem. 270, 2735–2740. Buckland, R.J., Watt, D.L., Chittoor, B., Nilsson, A.K., Kunkel, T.A., and Chabes, A. (2014). Increased and imbalanced dNTP pools symmetrically promote both leading and lagging strand replication infidelity. PLoS Genet. 10, e1004846.

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