Abstract

Next-generation sequencing (NGS) of single cells and micro-dissected tissues has demonstrated that the number of somatic mutations in normal cells increases linearly with age. The majority of somatic mutations are single base substitutions (SBSs). NGS has revealed the mutagenic effect of various metabolic and environmental genotoxic agents on different cells, such as normal colonic epithelial cells, hematopoietic cells, hepatocytes, and neurons. NGS has also uncovered the clonal expansion of cells in normal tissues of the elderly driven by oncogenic mutations. There are two main mechanisms for the generation of mutations, namely, replication errors and DNA damage, with both being followed by inaccurate repair. NGS studies of normal tissues highlight the substantial contribution of the latter mechanism by revealing the significant accumulation of somatic mutations in mitotically inactive hepatocytes and neurons. This review describes the nature of spontaneously arising SBSs in normal tissues, the causes of mutagenesis, cellular pathways of the DNA damage response that suppress mutagenesis, as well as unsolved questions on the matter.

Preamble

DNA damage arises from both endogenous (spontaneous) and exogenous (environmental) sources to then drive mutagenesis. Previous studies have identified a wide array of exogenous chemicals that cause genetic alterations as well as their precise genotoxic mechanisms, owing to the development of sensitive genotoxicity testing. However, the characterization of spontaneous mutations has been technically challenging since normal cell populations harbor various clones with distinct mutational profiles. NGS technology has been employed for the identification of small numbers of unique SBSs in single cells, their expanded populations, as well as micro-dissected tissues. The more recently developed error-reduced NGS strategies have sequencing error rates much lower than 5 × 10–8 base, which is comparable to the spontaneous mutation frequencies of mammalian genomes (10–9–10–7 base per cell division). Through these advances, NGS has been employed to reveal the mutagenic effects of environmental genotoxic agents in a variety of cells, such as normal colonic epithelial cells, hematopoietic cells, hepatocytes, and neurons. Sequencing has also revealed the clonal expansion of cells in normal tissues that occurs with aging. Subclonal multi-tissue analyses of somatic mutations have offered major insights into cell lineage commitment during embryonic development as well as into tissue-specific aspects of mutagenesis. This review summarizes basic concepts, introduces new insights, and discusses unresolved questions on the nature of mutations in normal tissues, the causes of mutagenesis, and cellular pathways that suppress mutagenesis.

The nature of mutations

Classification of mutations

Mutations are permanently transmissible changes in the amount and structure of genomic DNA in cells. These changes may involve a single base, gene, a block of genes, and whole chromosomes. In general, mutations include single base substitutions (SBSs), base deletions and insertions, gene amplification, and chromosomal rearrangements. Large-scale mutations also include changes in the number of chromosomes, resulting in aneuploidy (OECD, 2010).

SBS signatures were previously defined based on 96 mutation types. There are only six different possible substitutions that can occur between DNA bases: C>A, C>G, C>T, T>A, T>C, and T>G. These six nucleotide substitution types are further stratified based on the nucleotides immediately adjacent to the affected base pair, leading to the spectra of 96 mutation types (Fig. 1A) (Alexandrov et al., 2013, 2020; Nik-Zainal et al., 2012). Mutational signatures are defined based on the 96 mutation types and may arise as a result of multiple mutagenic processes, including the infidelity of DNA replication, exogenous or endogenous mutagen exposure, enzymatic modification of DNA, and defective DNA repair. Mutagenic signatures are presented in Fig. 1B. Mutational signature reference data are publicly available in the Catalogue of Somatic Mutation In Cancer (COSMIC) database (Alexandrov & Stratton, 2014; Alexandrov et al., 2013; Forbes et al., 2017) (see https://cancer.sanger.ac.uk/signatures/). Specific mutational signatures link endogenous and exogenous DNA damage and repair mechanisms (Table 1).

Mutational signatures. A 96 single base substitution (SBS) types. Red letters indicate the mutated nucleotides of the six substitution subtypes: C to A (the leftmost two columns), C to G (the third and fourth columns from the left), C to T (the fifth and sixth columns), T to A (the seventh and eighth columns), T to C (the ninth and tenth columns), and T to G (the rightmost two columns). These six SBSs are further subdivided, depending on the nucleotides immediately 5′ and 3′ to each mutated nucleotide. This subdivision generates all together 96 possible mutation types (six types of substitution × four types of 5′ base × four types of 3′ base). B Examples of mutation signatures, SBS1 and SBS5, which are known to be ‘clock’ mutations

Mutations occurring in the coding region of genes are classified depending on their effect on the functionality of protein products. This division includes silent mutations, missense mutations, nonsense mutations, and frameshift mutations, which result from nucleotide deletions and insertions. Mutations occurring in splicing signals often completely disrupt gene function. There is a 50% probability that loss of heterozygosity (LOH) will inactivate the function of affected genes with heterozygous mutations. LOH results from mutations in the intact allele, heteroallelic recombination between homologous chromosomes, and abnormal chromosome segregation in the M phase (Fig. 2). LOH in tumor suppressor genes with heterozygous mutations leads to clonal expansion and increases the risk of cancer. Clonal expansion of blood cells is commonly seen with age, increases the risk of leukemia, and is caused by copy-neutral loss of heterozygosity (CN-LOH) (Fig. 2, right bottom) (Loh et al., 2020; Terao et al., 2020). A typical example of LOH-dependent carcinogenesis is the presence of mutations in one of the two RB allelic genes. Carriers suffer from retinoblastoma with an extremely high penetrance due to the frequent loss of the intact RB gene (Knudson, 1971). LOH is also responsible for Hereditary Breast and Ovarian Cancer (HBOC) syndrome in women having mutations in the breast cancer gene 1 (BRCA1) and BRCA2 genes (see https://www.cancer.gov/about-cancer/causes-prevention/genetics/brca-fact-sheet). LOH also accounts for enhanced carcinogenesis in carriers of ATM and TP53 mutations (see “DNA damage checkpoints”) (Huang et al., 2018).

Schematic views showing four types of LOH. Assume a locus heterozygous for a wild-type allelic gene (marked with +) and a mutant allelic gene (marked with *). LOH can result from the loss-of-function mutation of the wild-type gene (left top), a long deletion (left bottom), and whole chromosome loss (right top). Recent NGS studies revealed copy-neutral loss of heterozygosity (CN-LOH) (right bottom). The molecular mechanism of generating CN-LOH is unknown

Origin of SBSs

SBSs are generated de novo during germ cell development as well as in somatic cells. NGS technology allows for the unbiased systematic investigation of mutations throughout the whole genome in large populations of people. Such investigations have revealed mutations present at the population level (see “Germline mutations”), mutations newly generated during germ cell development (see “De novo mutations (DNMs)”) (Goldmann et al., 2019), somatic cell mutations (see “Somatic mutations”), as well as the multi-step process of oncogenesis from normal tissues to malignant cancers (see “Multi-step oncogenesis”).

Germline mutations

Germline mutations are distinct from somatic mutations and are inherited by every somatic and germline cell of the offspring. An open database, gnomAD (Collins et al., 2020; Karczewski et al., 2020) (https://gnomad.broadinstitute.org/), stores germline mutations detected in a cohort of healthy individuals. Healthy individuals have an average of 140 germline truncating mutations in the coding region that significantly impair the function of encoded proteins (Lek et al., 2016). Surprisingly, healthy individuals harbor an average of 35 homozygous mutations that disrupt protein function, indicating that these mutated proteins are dispensable for embryogenesis and human health. Healthy people also have approximately 20 mutations in the germline that disrupt the function of encoded protein on one of the alleles, but these mutations are not homozygous, in which case they could cause prenatal lethality, developmental anomalies, and autosomal recessive diseases. In other words, healthy individuals harbor an average of 20 autosomal recessive mutations that would cause embryonic lethality and severe diseases if homozygous. Typical examples include mutations in the RB, BRCA1, and BRCA2 genes.

De novo mutations (DNMs)

DNMs are mutations that arise during germ cell development within a single generation and thus are not observed in the parents. DNMs act as drivers of evolution and are a major cause of genetic disease. A typical example is Down syndrome, caused by trisomy of chromosome 21, which develops as a result of missegregation during oocyte meiosis. NGS of parent–offspring trios allows for accurately measuring the frequency of DNMs in humans (Koboldt et al., 2013). The number of DNMs in offspring is positively correlated with the age of parents at conception, and thus the children of elderly parents more frequently suffer from conditions, such as intellectual disability, autism, and epilepsy (Krupp et al., 2017; McRae et al., 2017; Taylor et al., 2019; Veltman & Brunner, 2012; Wiener-Megnazi et al., 2012). Maternal and paternal nucleotide substitutions per year of aging have been estimated at 0.4 and 1.7 × 10–8, respectively (Goldmann et al., 2016, 2019; Kong et al., 2012; Rahbari et al., 2016). The de novo mutation rate of C57BL/6 laboratory mice is 0.54 × 10–8 per nucleotide per generation (Uchimura et al., 2015).

The paternal nucleotide substitution rate is several times higher than the maternal one, probably due to differences between sperm and oocyte development. During oogenesis, primordial germ cells (pGCs) are generated and enter meiosis I prior to the birth of female babies, subsequently completing it during ovulation to differentiate into mature oocytes. Thus, meiosis I of individual oocytes takes 15–50 years, with oocyte proliferation halted for extended periods. In contrast to the prenatal termination of oocyte generation, sperm precursors undergo active genome replication from puberty until approximately 50 years of age. Spermatogonia start cell division after birth and maintain it to supply mature sperm over an extended period. Accordingly, the number of DNA replication cycles necessary for preparing fertilization-ready gametes is greater in spermatogenesis than in oogenesis. Thus, more paternal SBSs are generated during aging relative to maternal ones. The precise mechanisms underlying maternal DNMs remain to be elucidated (Goldmann et al., 2019).

In a recently reported cohort study (Kaplanis et al., 2021), 12 hypermutated individuals were identified from over 20,000 parent offspring sequenced trios, with a two to sevenfold increase in the number of de novo single nucleotide variants (SNVs) in the genome. Both genetic and environmental exposures (e.g. chemotherapeutics) contributed to germline mutation rate variation. They found germline mutation rates were increased by the defects in DNA repair genes, XPC and MPG were identified from paternal mutator variants in the study. However, defects in DNA repair pathways do not always behave similarly in the soma and the germline, they found somatic mutator gene MBD4 did not have a detectable effect in the germline ((Kaplanis et al., 2021).

Somatic mutations

Somatic mutations occur in small groups of cells and even in single cells, depending on the timing and source of mutagenesis. NGS only recently elucidated the rate of somatic mutagenesis in various tissues. Methods for identifying mutations in normal tissues include sequencing DNA derived from (a) clonally expanded cells derived from single stem cells, (b) micro-dissected tissues derived from a small number of cells, and (c) single cells (Table 2). NGS revealed that somatic mutations increase linearly with age, similar to DNMs in germ cells (Goldmann et al., 2019). The mutation rate of actively cycling sperm precursors is approximately 27-fold lower than that of the colorectal epithelium (Moore et al., 2021). The comparison of various tissue samples across organs from the same individual indicated 2.4 mutations per cell division during the first few embryonic divisions (Table 2) (Coorens et al., 2021; Park et al., 2021). The rate of mutagenesis is subsequently reduced, presumably due to enhanced DNA repair activity.

NGS studies have demonstrated that cells in mitotically inactive tissues, such as hepatocytes and neurons, accumulate somatic mutations at rates similar to those in cells from mitotically active tissues, such as colon epithelial and hematopoietic cells (Table 2). Thus, somatic mutation rates vary only moderately between tissues, indicating that the mutagenesis of somatic cells is largely independent of division frequency (Abascal et al., 2021; Blokzijl et al., 2016). This finding was remarkable as DNA replication has long been considered the primary cause of somatic mutations due to DNA polymerase errors (Kunkel & Erie, 2015; Tomasetti & Vogelstein, 2015) and error-prone translesion DNA synthesis (TLS) (see the 5th paragraph of “Spontaneously occurring base damage”) (Quinet et al., 2021; Vaisman & Woodgate, 2020). The similar somatic mutation rates between mitotically inactive and active tissues suggest that endogenously arising DNA lesions followed by faulty repair are the significant source of somatic mutations. Further, the inaccurate repair of DNA damage accounts for the time-dependent, rather than cell cycle-dependent accumulation of somatic mutations (Abascal et al., 2021). The molecular mechanisms protecting cycling sperm precursors from the accumulation of somatic mutations and underlying time-dependent mutagenesis remain unresolved.

Multi-step oncogenesis

The constant accumulation of oncogenic mutations drives multi-step oncogenesis, which transforms normal tissues into malignant cancers (Ciriello et al., 2013; Futreal et al., 2004; Hanahan & Weinberg, 2011). Epidemiological studies indicate that the risk of developing cancer increases exponentially with age (Doll, 1971). That is, a twofold increase in age leads to an increase from 32 (25) to 256 (28) in the risk of developing cancer. This dramatic increase may be attributed to the rapid increases in mutation rates with age. However, the linear increase in somatic mutations with age (Table 2) suggests that the accumulation of five to eight oncogenic driver mutations in cells is more likely to account for the above-presented epidemiological data (Campbell et al., 2020). This assumption is also supported by a recent meta-analysis of 6904 tumor samples (Anandakrishnani et al., 2019).

The identification of genes whose mutant variants drive oncogenesis in individual malignant tumors is a major goal. Whole-genome NGS in normal and precancerous regions of the esophageal epithelia (Yokoyama et al., 2019), skin (Martincorena et al., 2015), colon (Lee-Six et al., 2019), ulcerative colitis tissue (Kakiuchi & Ogawa, 2021; Kakiuchi et al., 2020), hematopoietic cells (Collins et al., 2020; Jacobs et al., 2012; Jaiswal & Ebert, 2019; Karczewski et al., 2020; Saiki et al., 2021), bronchi (Yoshida et al., 2020), and urinary bladder (Lawson et al., 2020; Li et al., 2020) have identified driver mutations that promote multistep carcinogenesis. The number of mutations in the dividing cells of normal tissues is 1.14–1.37 mutations per division and is 4–100 times lower than that in adult malignant cells (Werner et al., 2020). The number of mutations is 14 times lower in childhood cancers than in adult cancers (Bandopadhayay & Meyerson, 2018; Gröbner et al., 2018; Kandoth et al., 2013; Ma et al., 2018). A high mutation frequency is associated with loss-of-function mutations in DNA damage response genes encoding p53, DNA-dependent protein kinase, mismatch repair factors (MSH2, MSH6, and PMS2), DNA polymerase ε (Polε), and DNA polymerase Q (PolQ) in adult and childhood cancers (Bandopadhayay & Meyerson, 2018; Gröbner et al., 2018; Kandoth et al., 2013).

The cause of spontaneous mutagenesis

Non-dividing cells in mitotically inactive tissues accumulate considerable somatic mutations, as discussed in “Somatic mutations”, indicating the presence of DNA replication-independent mutagenesis in addition to mutagenic mechanisms dependent on replication (Fig. 3). Replication-dependent mutagenesis is induced by the activity of replicative DNA polymerases (Fig. 3A) and translesion DNA synthesis (TLS) polymerases (Fig. 3B, C). Replication-independent mutagenesis is presumably initiated by DNA damage and subsequent inaccurate DNA repair. This chapter summarizes replication errors and DNA damage, while the next chapter describes DNA repair pathways.

Mechanism for the mutagenesis during DNA replication. A Replicative DNA polymerases occasionally misincorporate nucleotides; for example, G opposite to A. MMR repairs the resulting G: A mispair and inserts T opposite to A. If MMR fails to repair the G: A mispair, the second DNA replication causes C to A substitution. B Translesion DNA synthesis (TLS) past a thymidine dimer, UV damage. The vast majority of the DNA lesions block the DNA synthesis by replicative DNA polymerases d and e (Pol d and Pol e). TLS polymerases insert a few nucleotides, shown as ‘NN’ in red, opposite DNA damage in an error-prone manner. Replicative DNA polymerases then extend DNA synthesis. C Regulation of TLS by mono-ubiquitination of the clamp protein, PCNA. UV damage stops Pol d and Pol e, leading to the mono-ubiquitination of PCNA. Ubiquitinated PCNA provides the docking site for TLS polymerase, Pol h, which inserts a few nucleotides opposite DNA lesions. D The slippage of replicative DNA polymerases changes the copy number of short tandem repeats (STRs) such as CAG repeats. The slippage towards the opposite direction of DNA synthesis increases the copy number, resulting in the expansion of repeats. The slippage can decrease the copy number, leading to the contraction of repeats

Replication errors

The fidelity of DNA replication is primarily determined by the nucleotide selectivity and proofreading activity of replicative DNA polymerases as well as mismatch repair (MMR) machinery (Fig. 3A) (Ganai & Johansson, 2016). The replication error rate per division is once per 107 bases in yeast, Saccharomyces cerevisiae (St Charles et al., 2015), versus 1.14–1.37 mutations per 6 × 109 bases in normal human cells (Werner et al., 2020). Proofreading mechanisms and MMR remove misincorporated dNTPs, increasing the fidelity of DNA replication 250-fold in yeast (Lujan et al., 2012; Morrison & Sugino, 1994). MMR removes different types of mismatches with variable efficiency (St Charles et al., 2015). For example, C–T mismatches are corrected with 80 times greater efficiency than T–T mismatches. Thus, the variable efficiency of MMR is likely to determine SBS signatures in replicating cells. In addition to MMR, RecQ DNA helicase replication errors also arise when replicative DNA polymerases incorporate ribonucleotide triphosphates (rNTPs) into genomic DNA. The concentration of rNTPs is much higher than that of dNTPs in vivo (Nick McElhinny et al., 2010), and replicative DNA polymerases incorporate one rNTP for every 103–104 dNTPs (Ashour & Mosammaparast, 2021; McElhinny et al., 2010; Nick McElhinny et al., 2010). While most misincorporated rNMPs are removed by ribonuclease H2, rNTPs incorporated in the DNA can trap DNA topoisomerase I (TOP1), leading to the deletion of 2–5 nucleotides (Clark et al., 2011; Kim et al., 2011; Zimmermann et al., 2018).

Roughly 3% of the human genome is comprised of simple sequence repeats (Lander et al., 2001). Short tandem repeats (STRs) are defined by motifs with a length of six or fewer bases repeated many times (5–50), including repeats composed of (A)n, (CA)n, (TA)n, (CAG)n, and (CTG)n (Ellegren, 2004). STRs are unstable and prone to changes in copy numbers with a frequency as high as 10−4–10−3 per generation (Ellegren, 2004). The copy number change results from the slippage of replicative DNA polymerases during replication (Fig. 3D) (Viguera et al., 2001). WRN stabilizes TA-dinucleotide repeats in replicating cells (van Wietmarschen et al., 2020). Copy number changes can also occur independently of DNA replication, as the expansion of trinucleotide repeats is observed in non-dividing neurons and oocytes (Gonitel et al., 2008; Kovtun & McMurray, 2001; Kovtun et al., 2000). However, the molecular mechanism underlying the expansion of quiescent cells remains unclear (Polyzos & McMurray, 2017).

Spontaneously occurring base damage

Approximately 70,000 DNA lesions per cell are generated daily via hydrolysis, alkylation, and oxidation of nucleotides as well as the subsequent generation of single-strand breaks (SSBs) (Lindahl, 1993; Lindahl & Barnes, 2000; Tubbs & Nussenzweig, 2017). The base excision repair (BER) pathway repairs base damage, with SSBs being formed as intermediate repair substrates (Fig. 4A, B). DNA replication past unrepaired damaged nucleotides on template strands causes mutations (Fig. 4A–C).

Molecular mechanisms for spontaneous and UV-induced mutagenesis. A Spontaneous deamination of 5-methylcytosine. Its deamination generates T resulting in a T: G mispair. BER involving TDG, APE, and Pol b changes T to C. B Oxidation of G by ROS generates 8-oxoguanine, and BER involving OGG1, APE, and Pol b repairs the damage. If replication occurs before BER, replicative DNA polymerases often incorporate A opposite to 8-oxoguanine. An MMR factor, MUTYH, removes A and restores an 8-oxoguanine: C pair. If the second round of replication occurs before MMR, it leads to G to T base substitution. C UV irradiation generates crosslinks between two neighboring pyrimidines. NER involving XPA, XPF, and XPG removes the resulting cytosine-cytosine photodimers. Replicative DNA polymerases are unable to undergo DNA synthesis past UV lesions. TLS polymerases then erroneously perform DNA synthesis and insert, for example, two adenines opposite to cytosine-cytosine photodimers. NER and the second round of replication using the AA-containing template strands cause C to T base substitution at UV-damaged CC dinucleotides

In neurons, mutation hotspots are localized at DNase-I-hypersensitive sites (Dileep & Tsai, 2021; Lodato et al., 2015; Wu et al., 2021), presumably because genomic DNA in euchromatin is more susceptible to base damage than that in heterochromatin. Similarly to ROS, formaldehyde is generated from various biochemical reactions and environmental factors (Burgos-Barragan et al., 2017). Formaldehyde considerably damages genomic DNA, particularly in people lacking the appropriate formaldehyde-detoxifying enzymes (Dingler et al., 2020; Garaycoechea et al., 2018). Acetaldehyde also damages DNA, with alcohol intake increasing esophageal cancer risk (Matejcic et al., 2017). APOBEC family enzymes damage both viral genomes and human genomic DNA through cytidine deaminase activity, causing C>T and C>G mutations at TCN sequences of SBS2 and SBS13 in normal tissues (Table 1).The activation-induced deaminase (AID) catalyzes cytidine deamination at immunoglobulin V (Ig V) segments and is responsible for their hypermutation (Di Noia & Neuberger, 2007). Unlike APOBEC enzymes, AID causes hypermutation at A:T pairs and C:G pairs within Ig V segments, yet the molecular mechanisms underlying mutagenesis at A:T pairs remain unknown.

The CpG>TpG transition occurs during DNA replication as well as independently of the replication process. 75% of CpG cytosines are methylated in human cells, and deamination of 5-methylcytosine generates thymine (Fig. 4A). If the resulting T:G mismatch is left unrepaired, DNA replication over the thymine leads to CpG>TpG transition mutations (Table 1, SBS1) (Abascal et al., 2021; Fryxell & Zuckerkandl, 2000; Lindahl, 1993). CpG>TpG transition mutations accumulate 2.5 substitutions per year in human post-mitotic neurons, indicating that this transition can occur independently of DNA replication (Abascal et al., 2021). The mechanism underlying mutagenesis outside the S phase remains an open question, and the very frequent CpG>TpG transition likely stems from inefficient recognition of the T:G mismatch (Tubbs & Nussenzweig, 2017). SBS1 is the second most common base substitution signature after SBS5 (Abascal et al., 2021; Moore et al., 2021). CpG>TpG transitions account for the majority of C>T mutations, reflecting the inherent mutability when cytosine is methylated at CpG sequences (Lindahl, 1993). In addition to 5-methylcytosine, hydroxymethylated cytosines are associated with increased C to G mutation rates (Supek et al., 2014).

NGS studies suggest that ROS only modestly contribute to time-dependent mutagenesis. ROS generated via cellular respiration have been proposed as drivers of aging and oncogenesis. Daily, ROS induce approximately 2800 8-oxoguanine (8-oxoG) alterations per cell (Tubbs & Nussenzweig, 2017). 8-oxoG can then mispair with adenine, and replicative DNA polymerases often misincorporate adenine opposite to 8-oxoG, resulting in G>T substitutions (Fig. 4B). 8-oxoG is widely believed to be a primary driver of mutagenesis. However, NGS has revealed that mutations resulting from oxidative DNA lesions, including 8-oxoG-dependent SBS18 (Table 1), do not significantly increase with age neither in genomic nor in mitochondrial DNA (Helleday et al., 2014; Kennedy et al., 2013). These data highlight the efficient repair of oxidative damage.

Base damage is converted to SBSs during DNA replication through the TLS mechanism. DNA lesions on template strands often block DNA synthesis by replicative DNA polymerases (Figs. 3B, C, 4C). The DNA damage tolerance (DDT) mechanism restores the progression of DNA replication forks without repairing these lesions. Instead, DDT employs TLS (Fig. 3B, C), template switching, and priming DNA synthesis via PrimPol (Ashour & Mosammaparast, 2021; Quinet et al., 2021; Wong et al., 2021) to proceed over lesions. TLS is carried out by DNA polymerases Pol η, Pol ι, Pol κ, Rev1, and Pol ζ. Pol β, Pol λ, Pol θ, Pol ν, and PrimPol are also involved in TLS (Bainbridge et al., 2021; Goodman & Woodgate, 2013). These polymerases can insert a few nucleotides opposite to various lesions, followed by a restart of DNA synthesis by replicative DNA polymerases (Fig. 3B). TLS polymerases have a fidelity that is several orders of magnitude lower than that of replicative DNA polymerases during the copying of undamaged DNA substrates (McCulloch & Kunkel, 2008). Yeast Pol ζ is responsible for over 95% of UV-induced mutagenesis and 50–75% of spontaneously arising mutations (Lawrence & Maher, 2001). The contribution of TLS to spontaneous somatic mutations in cycling mammalian cells is unclear because of the complex functional interactions between TLS polymerases. TLS DNA polymerases also play a role in excision DNA repair (Kawamoto et al., 2005; Ogi et al., 2010; Yoshimura et al., 2006). Of note, their involvement in somatic mutations in non-dividing cells remains unresolved.

Spontaneously occurring SSBs

An estimated 55,000 SSBs arise per day in each cell (Tubbs & Nussenzweig, 2017). The common triggers of SSBs are endogenous ROS. Unlike alkylating agents, such as methyl methanesulfonate (MMS), the exposure of cultured cells to H2O2 efficiently induces SSBs even on ice (Demin et al., 2021), indicative of direct cleavage of DNA strands by H2O2. SSBs occur during BER and as a consequence of erroneous enzyme catalysis. The most relevant enzyme in this regard is TOP1, which is essential for every transcription and replication event. TOP1 constantly repeats the cycle of SSB formation immediately followed by its religation. When TOP1 cleaves DNA to relieve torsional stress, broken ends rotate along the other intact strand, and TOP1 religates the SSBs only after stress relief. TOP1 cannot efficiently religate if it cleaves DNA sites near DNA damage, including SSBs, UV damage, ribonucleotides, abasic sites, DNA-RNA hybrids (R-loops), and G-quadruplex (G4 DNA) because these lesions dislocate the two ends of the breakage (Jakobsen et al., 2019; Kim et al., 2011; Pommier et al., 2016; Saha et al., 2020). The TOP1 trap at rNTPs embedded in DNA can lead to 2–5-nucleotide deletions (Clark et al., 2011; Kim et al., 2011; Zimmermann et al., 2018). Nonetheless, it is generally believed that SSBs are accurately repaired due to the presence of the other intact strand.

Spontaneously occurring DSBs in non-dividing cells and during DNA replication

DNA double-strand breaks (DSBs) cause indels (short deletions and insertions), copy number variation (long deletion and gene amplification), inversion, and chromosome translocation. Lethal DSBs are generated by environmental factors, such as X-irradiation and endogenous cellular processes. Recent studies have uncovered the role of DNA topoisomerase II (TOP2) in spontaneously occurring DSBs in non-dividing cells (Fig. 5A) (Morimoto et al., 2019; Tubbs & Nussenzweig, 2017). There are two TOP2 isoforms, TOP2A and TOP2B, which are essential for transcriptional elongation, particularly in long genes (Austin et al., 2018; Pommier et al., 2016). TOP2 enzymes resolve DNA catenanes by generating transient ‘gated’ DSBs, where TOP2 homodimers are covalently bound to the two ends of individual DSBs (Fig. 5A). ‘Abortive’ catalysis seems to occur frequently, leaving gated DSBs unsealed (Bunch et al., 2015; Gómez-Herreros et al., 2014; Haffner et al., 2010; Hoa et al., 2016; Ju et al., 2006; Madabhushi et al., 2015; Sasanuma et al., 2018; Williamson & Lees-Miller, 2011). TOP2-dependent DSBs may represent the recurrent DSBs seen in long transcribed gene bodies in neural progenitor cells (Wei et al., 2016) and cause over one megabase-scale de novo copy number variation in human neurons (McConnell et al., 2013). TOP2-dependent DSBs may be responsible for the unusual enrichment of indels in highly expressed genes (Abascal et al., 2021).

Two significant sources of double-strand DNA breakage, the abortive catalysis of TOP2 and replication blockage at damaged template strands. A TOP2 continuously generates a considerable number of double-strand breaks (DSBs) called TOP2 cleavage complexes (TOP2ccs), where TOP2 covalently binds to 5′ end of DSBs (Step 1). Intrinsic ligation activity of TOP2 seals DSBs (Step 2). TOP2 often fails to religate TOP2ccs, called abortive catalysis of TOP2 (Step 3), generating stalled TOP2ccs (Step 4). Their religation is carried out by the removal of 5′ TOP2 adducts (Step 5) followed by canonical nonhomologous end-joining (c-NHEJ) (Step 6). B When replication forks encounter an SSB (Step 1), it is converted to a breakage called one-end breakage (Step 2). Homologous DNA recombination (HR) repairs breaks using the other sister chromatids as a template (Steps 3–5). C The vast majority of base lesions stop DNA replication (Step 1), and the replication blockage is released by HR (Steps 2–5) as well as TLS. Note that neither HR nor TLS repairs the DNA lesions that block the progression of replication forks. HR releases replication blockage in an error-free manner while TLS is error-prone

DNA replication forks stall at SSBs on template strands, generating one-end DSBs in one of the two sister chromatids (Fig. 5B). Homologous DNA recombination (HR) accurately repairs DSBs using the other sister chromatid as a template (Fig. 5B). Similarly to TLS (Fig. 3B, C), HR releases replication blockage at damaged nucleotides without repairing the damage (Fig. 5C). The complete loss of HR activity generates multiple DSBs and immediately kills dividing cells (Sonoda et al., 1998). The number of spontaneously occurring DSBs is much lower than that of spontaneously arising SSBs, with individual cells generating approximately 25 DSBs per day, yet DSBs are considerably more dangerous than SSBs (Tubbs & Nussenzweig, 2017). Active transcription often generates R-loops, and their collision with replication forks induces DSBs (García-Muse & Aguilera, 2019). Prolonged replication blockage at damaged nucleotides can also cause DSBs due to endonucleolytic cleavage by Mus81 (Ashour & Mosammaparast, 2021).

Inaccurate repair of DNA damage in spontaneous mutagenesis

Excision repair

Cells have three excision repair pathways, BER (Fig. 4A, B), nucleotide excision repair (NER) (Fig. 4C), and MMR (Figs. 3A, 4B) (Lindahl et al., 1997). BER and MMR are mainly responsible for correcting errors generated via spontaneous mutagenesis, while NER is involved in the repair of damage induced by exogenous chemicals and UV irradiation. BER is essential for genomic stability in mammalian cells (see “Spontaneously occurring base damage”). Mice deficient in key BER enzymes, such as apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase β (Pol β) (Fig. 4A, B), are embryonic lethal and die immediately after birth, respectively (Gu et al., 1994; Ludwig et al., 1998).The severe phenotype of BER-deficient animals contrasts with humans deficient in NER and MMR, who develop normally. MMR defects cause carcinogenesis in the colon epithelium in humans due to the accumulation of point mutations and changes in the copy number of STRs during DNA replication (see “Replication errors”) (Peltomäki et al., 2020). If excision repair fails to remove base damage before DNA replication, error-prone TLS past damaged template strands converts DNA damage to mutations (see “Spontaneously occurring DSBs in non-dividing cells and during DNA replication” and Fig. 3B, C).

BER constantly repairs large numbers of spontaneously arising damaged bases in both mitotically active and inactive tissues. Thus, inaccurate BER events are most likely to cause somatic mutations that accumulate linearly with age in mitotically inactive tissues. BER is initiated via the excision of damaged DNA bases by DNA glycosylases generating abasic sites, and human cells possess 11 glycosylases (Svilar et al., 2011). Increased expression of DNA glycosylases can enhance spontaneous mutagenesis in bacteria, yeast, and mammalian cells (Fu et al., 2012). Repair synthesis in BER is performed by replicative DNA polymerases δ and ε (Pol δ and Pol ε), Pol β, Pol λ, as well as Pol Θ (Braithwaite et al., 2010; Yoshimura et al., 2006) (Fig. 4A, B). However, the relative contribution of these polymerases in normal tissues is unclear. The latter three enzymes lack proofreading activity, and their fidelity is a few orders of magnitude lower than that of Pol δ and Pol ε. The actual mutation frequency of BER in vivo remains poorly understood.

The loss of MMR promotes tolerance, rather than sensitization, to alkylating agents. The molecular mechanisms underlying this tolerance are not yet fully understood. One identified mechanism is that alkylating agents form O6-methylguanine (O6meG), and DNA replication past the damaged nucleotide generates O6meG:T mispairs. MMR recognizes these, initiating futile cycles of DNA resection and the restoration of O6meG:T mispairs, eventually leading to mutagenic repair, DNA breakage, and apoptosis (Cejka & Jiricny, 2008; Fu et al., 2012; Mojas et al., 2007). It is not yet clear why BER cannot efficiently remove O6meG from the O6meG:T mispair. The functional interaction between the BER and MMR is discussed in the 4th section. BER can repair UV-induced lesions in NER-deficient cells when UV lesions irreversibly trap stable TOP1-SSB complexes near UV damage, with the resulting SSBs activating BER (Saha et al., 2020).

The contribution of BER, NER, and MMR to mutagenesis in mitotically inactive tissues has not yet been clarified. The neurodegeneration observed in mice deficient for XRCC1, a key BER factor, is primarily attributable to the depletion of NAD+, a substrate for poly (ADP ribose) polymerase, rather than the accumulation of mutations (Demin et al., 2021; Hoch et al., 2017). In NER-deficient patients, who have a mild neurodegenerative phenotype from puberty, neurons exhibit a several times greater accumulation of somatic mutations (Lodato et al., 2018). In summary, future studies are required to determine whether BER generates mutations in post-mitotic cells. Another unsolved question is whether the loss of MMR increases de novo mutations in non-dividing cells.

SSB and DSB repair

BER repairs smaller SSBs with extremely high efficiency within 60 min after pulse exposure to H2O2 (Hoch et al., 2017). Ligase-dependent rejoining of SSBs requires the preceding restoration of a phosphate group at their 5′ end and a hydroxy group at their 3′ end. Various enzymes are involved in the restoration, including polynucleotide kinase-phosphatase (PNKP) (Kalasova et al., 2020; Plo et al., 2003), the lyase function of Pol β removing a 5′-deoxyribose phosphate (5′-dRP) moiety of SSBs (Beard & Wilson, 2019; Sobol et al., 1996), DNA-apurinic–apyrimidinic (AP) endonuclease I (APE1, APEX1) (Caldecott, 2008), AP endonuclease 2 (APE2, APEX2) (Álvarez-Quilón et al., 2020; Li et al., 2019), aprataxin (APTX, AOA1) (Ahel et al., 2006; El-Khamisy et al., 2009; Grundy et al., 2013), and tyrosyl-DNA phosphodiesterase 1 (TDP1, SCAN1) (El-Khamisy et al., 2005; Sun et al., 2020a; Zeng et al., 2012). Homozygous mutations in the APTX, TDP1, and PNKP genes cause neurodegenerative diseases (Date et al., 2001; Moreira et al., 2001; Shen et al., 2010; Takashima et al., 2002). Further, the repair of TOP1-SSB complexes requires the coordinated action of TDP1 and PNKP (Sun et al., 2020b). No report has provided compelling evidence for the accumulation of mutations or defective SSB repair as the cause of the diseases.

Mammalian cells employ three DSB repair pathways, namely, HR, canonical non-homologous end-joining (c-NHEJ), and alternative end-joining (alt-EJ) (Ceccaldi et al., 2016). HR repairs DSBs when intact sister chromatids are available as template DNA (Fig. 5B, C) in the S to G2 phases, while NHEJ and alt-EJ operate throughout the cell cycle as well as in post-mitotic cells (Ceccaldi et al., 2016). alt-EJ has not yet been thoroughly characterized, operating as a backup to abrogated HR and c-NHEJ, with major contribution to the formation of chromosomal translocations (Bennardo et al., 2008; Ranjha et al., 2018). Pol Θ seems to play a key role in alt-EJ independently of canonical c-NHEJ (Ceccaldi et al., 2015; Saito et al., 2017).

DSBs generated during DNA replication are accurately repaired exclusively via HR between broken sister chromatids and intact chromatids (Dias et al., 2021; Thomas & Pommier, 2019) (Fig. 5B). In contrast, c-NHEJ plays a dominant role in the repair of TOP2-dependent DSBs (Maede et al., 2013) (Fig. 5A). c-NHEJ requires the removal of blocking adducts (Akagawa et al., 2020; Chappell et al., 2002). TOP2 adducts and 5′-blocking adducts are removed via TDP2 and MRE11 endonucleases (Deshpande et al., 2016; Hoa et al., 2016; Ledesma et al., 2009; Paull, 2018). It remains unclear whether MRE11-dependent repair can accurately repair DSBs covalently associated with TOP2 (Zagnoli-Vieira & Caldecott, 2020). DSBs generated via endogenous sources and ionizing-radiation (IR) generally carry blocking adducts at their ends and are termed ‘dirty’ DSBs (Woodbine et al., 2011). A recent study reported that ARTEMIS, EXD2, EXO1, and MRE11 nucleases contribute to ‘cleaning up’ dirty ends in preparation for subsequent c-NHEJ (Akagawa et al., 2020). It is unclear whether the cleaning up followed by c-NHEJ results in the deletion of nucleotides.

DNA damage checkpoints

DNA damage checkpoint (DDCs) sense DNA damage and initiate signal transduction through a kinase cascade, leading to transient cell cycle arrests at the G1/S transition, S phase, and G2/M transition (Paulovich & Hartwell, 1995; Weinert & Hartwell, 1988). The DDC is fully activated by a small number of unrepaired DSBs, HR intermediates, and stalled replication forks, providing cells with time to repair DNA (Lecona & Fernandez-Capetillo, 2018; Paulovich & Hartwell, 1995; Waterman et al., 2020; Weinert & Hartwell, 1988). During transient cell cycle arrest at the G1/S checkpoint, cells repair DNA damage to prevent the collapse of replication forks at broken DNA. DSB repair at the G2/M checkpoint prevents long deletions distal to DSBs and whole-chromosome loss during mitosis. In addition to cell cycle arrest, DDC promotes the HR-dependent repair of DSBs, thereby suppressing error-prone c-NHEJ when DSBs are generated due to DNA replication at broken template strands (Balmus et al., 2019). In cycling cells, the above mechanisms suppress DSB-induced mutagenesis, including deletion, chromosome translocation, and chromosome loss. If a single DSB is left unrepaired within a time window, activated DDCs eventually trigger apoptosis and eliminate cells carrying unrepaired DSBs from the body (Trenner & Sartori, 2019). The key players in DDC signaling are the ATM kinase and its substrate, the TP53 transcription factor (Banin et al., 1998; Lee & Paull, 2021). ATM and TP53 prevent oncogenesis by suppressing DSB-induced mutagenesis (Bailey et al., 2018; Honma & Hayashi, 2011), and carriers of ATM and TP53 mutations are predisposed to cancer (Huang et al., 2018). Although TP53 suppresses DSB-induced mutagenesis, TP53 does not seem to suppress SBSs, as indicated by TP53-deficient mice not showing a significant increase in SBSs neither in somatic tissues nor in the germline (Burr et al., 2005; Sands et al., 1995).

Unsolved questions and future prospect

Previous studies have examined the molecular mechanisms of mutagenesis by exposing cycling cells to exogenous DNA damage. Only recently did NGS studies reveal the accumulation of somatic mutations in normal tissues. Nevertheless, the mechanisms underlying mutagenesis in normal tissues are poorly understood, particularly in post-mitotic cells. Mutagenesis in G0/G1 phases is considered to result from erroneous BER of endogenous DNA damage, considering that BER repairs approximately 70,000 DNA lesions per cell daily (Lindahl, 1993; Lindahl & Barnes, 2000; Tubbs & Nussenzweig, 2017). Mutations occur at a rate of less than one per week in humans (Table 2), and it is a formidable challenge to understand the mechanism underlying such extremely rare events of mutagenesis. The number of SBSs in maternal and paternal germ cells is only 0.4 and 1.7, respectively, per year during aging (Goldmann et al., 2016, 2019; Kong et al., 2012; Rahbari et al., 2016) and over ten times lower than that in somatic cells (Table 2). The mechanism underlying such a low frequency in germ cells represents an essential topic for future studies.

B lymphocytes undergo programmed SBSs specifically at the variable region of the Ig V gene, increasing the affinity of the immunoglobulin to antigens. This Ig V hypermutation is initiated by the deamination of dC residues via AID, generating dU:dG mispairs (Di Noia & Neuberger, 2007; Muramatsu et al., 2000). BER and MMR are complementary in processing dU:dG mispairs and are required for Ig V hypermutation (Rada et al., 2004). The removal of dU by uracil-DNA glycosylase and TLS over the resulting abasic site causes hypermutation at dC:dG pairs (Nakahara et al., 2009; Sale, 2013). Overall, the molecular mechanism of MMR-dependent mutagenesis at Ig V is poorly understood. Another unsolved question is the molecular mechanism underlying hypermutation at dA:dT pairs. Addressing these questions in the future may further our understanding of the molecular mechanisms underlying mutagenesis, which involve BER and MMR in normal tissues.

It is well established that the accumulation of SBSs cause carcinogenesis. However, it is unclear whether accumulated SBSs promote aging and progressive neurodegenerative diseases as evidenced by the phenotype of MMR-deficient people. MMR-deficiency neither acerates aging nor cause neurodegenerative diseases although the number of newly generated SBSs per division increases nearly 100 times (Peltomäki et al., 2020). Thus, it is an issue of future study to clarify whether a few times increase in the number of SBSs (Table 2) causes neurodegenerative symptom in NER-deficient CS an d XP patients (Lodato et al., 2018). Likewise, patients deficient in BER factors, APTX, TDP1, and PNKP, suffer neurodegenerative diseases (Date et al., 2001; Moreira et al., 2001; Shen et al., 2010; Takashima et al., 2002), the contribution of increased SBSs to the disease is an important question for future research.

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Acknowledgements

This work is supported by JSPS KAKENHI (16H12595 and 16H06306) (to ST), and by the JSPS Core-to-Core Program, A. Advanced Research Networks (to ST), and by the Major Program of the National Natural Science Foundation of China, grant number 41991314.

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Affiliations

1. Shenzhen University School of Medicine, Guangdong, Shenzhen, 518071, China

   Shunichi Takeda

2. School of Medicine, Kyoto University, Yoshida Konoe, Kyoto, 606-8501, Japan

   Shunichi Takeda

3. Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China

   Yang Luan

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Correspondence to Shunichi Takeda or Yang Luan.

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Takeda, S., Luan, Y. Nature of spontaneously arising single base substitutions in normal cells. GENOME INSTAB. DIS. 2, 339–357 (2021). https://doi.org/10.1007/s42764-021-00056-9

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• Received19 October 2021

• Revised09 November 2021

• Accepted17 November 2021

• Published26 November 2021

• Issue DateDecember 2021

• DOIhttps://doi.org/10.1007/s42764-021-00056-9

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Keywords

• Spontaneous mutagenesis

• Error-reduced NGS technology

• Replication errors

• Inaccurate repair of DNA damage

• de novo mutation