Biological functions of translesion synthesis proteins in vertebrates

DNA Repair 2 (2003) 1075–1085

Mini review

Biological functions of translesion synthesis proteins in vertebrates Jacob G. Jansen, Niels de Wind∗ Department of Toxicogenetics, Leiden University Medical Center, Wassenaarseweg 72, 2300 RA Leiden, The Netherlands Received 19 June 2003; accepted 20 June 2003

1. Introduction Shortly after exposure to a low dose of ultraviolet (UV) light, vertebrate cells in S-phase show the appearance of small nascent DNA strands, suggesting an accumulation of daughter strand gaps due to stalled replication forks at UV lesions [1]. DNA of normal size is synthesised at later times, despite the presence of persistent DNA lesions that arrest replicative polymerases. This phenomenon, labelled with the misleading term ‘post-replication repair’, is abolished in cells of patients suffering from the autosomal recessive disorder Xeroderma pigmentosum variant (XP-V) and has been used to identify the gene that is defective in XP-V [2–5]. This gene encodes for Polymerase ␩ (pol␩), a member of the evolutionary conserved Y-family of DNA polymerases [6] that also includes Polymerase ␬ (pol␬), Polymerase ␫ (pol␫) and Rev1. In vitro assays show that many of these polymerases have translesion synthesis (TLS) activity, i.e. they insert nucleotides opposite damaged bases in DNA, resulting in a ‘mismatch’ that is efficiently extended by Polymerase ␨ (pol␨), a heterodimer of Rev3 and Rev7. Thus, these polymerases may resolve arrested replication forks.

∗ Corresponding author. Tel.: +31-71-5271607; fax: +31-71-5276173. E-mail address: n.de [email protected] (N. de Wind).

The biochemical characterisation of TLS polymerases has been the topic of recent reviews [7–10] and the role in vivo of Rev1, Rev3, Rev7 and Rad30 (pol␩) in Saccharomyces cerevisiae has been analysed thoroughly (reviewed in [7,11]). The last few years, however, several vertebrate cell lines and mouse models have been generated that allow an analysis of the in vivo function of TLS-associated proteins in higher eukaryotes. Apart from XP-V cells, these models have targeted disruptions in genes encoding the Rad6 and Rad18 upstream genes, in TLS polymerases [Rev1, Rev3, DinB1 (encoding pol␬)] or express antisense RNA (the upstream gene Mms2, Rev1, Rev3) to reduce endogenous expression of TLS genes (Table 1). In addition, mouse- and human-cell lines have been generated that over-express pol␬ [12,13]. This review will focus mainly on the cell biology of vertebrate TLS polymerases and their differential roles in cell cycle progression, cell survival and mutagenesis. Also recent studies on the regulation for TLS activity in vertebrate cells will be discussed.

2. Upstream proteins in lesion bypass Genetic studies in S. cerevisiae show that genes encoding TLS polymerases are epistatic to RAD6 and RAD18 (reviewed in [11]). Rad6-null mutants exhibit a pleiotropic phenotype, including defects in post-replication repair, in gene silencing and in

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Table 1 Vertebrate models for TLS polymerases and their phenotypes Polymerase

Rev3

Pol␩

Pol␫

Cell proliferation

Sensitivity

Inactivation

Chicken DT40 cells

Disruption

Low

Slow

Human fibroblasts Mouse ES cells

Antisense
BRCT

Normal Normal

Normal Normal

Mouse

Disruption

Mouse Human fibroblasts Human B cells

Antisense Antisense Antisense

Embryonic lethal Normal Normal Normal

Normal Normal

= (UV, BPDE)

Human cells

N-terminal truncation N-terminal truncation

Normal

Normal

↑↑↑ (UV + c)

Human Pol␬

Viability

Organism

C.A.

SCE

Mutability

Somatic hypermutation Frequency

↑↑↑ (UV, 4-NQO, H2 O2 , X-rays, CisPt) = (UV) ↑ (UV, 4-NQO, MNU)

↑↑ (UV)

= (4-NQO, H2 O2 )

↑↑ (UV)

= (UV)

↓↓↓

[28]

↓↓-↓↓↓ (UV) ↓↓ (UV)

[35] [JGJ + NdW]

↑↑↑ (NA-AAF)

[29–33] ↓↓

↑A-T; ↓G-C

↓↓ (UV, BPDE) ↓↓ = (UV + c)

= (UV + c)

↑↑ (UV)

↑↑ (UV)

[62] [36] [63] [37,42,43,49,50]

=

Reduced ↑↑ (UV) = (MMS, X-rays, ␥-rays, AAF, B[a]P, CisPt) ↑↑↑ (B[a]P) ↑ (UV) = (X-rays)

References

Spectrum

↑A-T; ↓ G-C

↑↑ (4-NQO)

[67]

Chicken DT40 cells

Disruption

Normal

Normal

[20] [20]

Mouse ES cells

Disruption

Normal

Normal

Mouse

Disruption

Normal

Normal

=

=

[38,39] [38] [39,71]

Human B cells Mouse

Disruption N-terminal truncation

Normal Normal

Normal Normal

↓↓↓ =

=

[72] [73]

↑↑↑ (B[a]P)

C.A.: chromosomal aberrations; SCE: sister chromatid exchanges; ↑: increase; ↓: decrease; =: similar; UV: ultraviolet; UV + c: UV + caffeine; 4-NQO: 4-nitroquinoline-1-oxide; H2 O2 : hydrogen peroxide; CisPt: cis-platin; MNU: methylnitrosourea; NA-AAF: N-acetoxy-acetylaminofluorene; AAF: N-2-acetylaminofluorene; BPDE: (±)-7␤,8␣-dihydroxy-9␣,10␣-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene; B[a]P: benzo[a]pyrene; MMS: methyl methanesulphonate; A-T: A-T base pairs; G-C: G-C base pairs; JGJ + NdW: Jansen and De Wind, unpublished results.

J.G. Jansen, N. de Wind / DNA Repair 2 (2003) 1075–1085

Rev1

Model

J.G. Jansen, N. de Wind / DNA Repair 2 (2003) 1075–1085

sporulation. Rad6- and rad18-null mutants are extremely sensitive to a wide variety of DNA damaging agents such as UV, X-rays, ␥-rays, 4-nitroquinoline oxide (4-NQO) and alkylating agents. In addition, these mutants show a hypomutable phenotype for UV and increased spontaneous mutagenesis, suggesting that DNA damage-dependent TLS is controlled by RAD6/RAD18. RAD18, a protein containing a RING-finger motif found in ubiquitin ligases, binds to single-stranded DNA and forms a complex with Rad6, a classical ubiquitin-conjugating enzyme [11]. In yeast, a specific lysine residue of the DNA polymerase clamp PCNA appears to be a target molecule for RAD6/RAD18-mediated mono-ubiquitination [14]. Multi-ubiquitination is performed by the putative ubiquitin ligase RAD5, which binds to RAD18, and by a heterodimer of UBC13 and MMS2, an ubiquitin-conjugating enzyme that is recruited to chromatin via RAD5 (and thus via RAD18). This PCNA modification appears to be specific for members of the RAD6 epistasis group and therefore could be important in the DNA damage response during S-phase. Indeed, a physical interaction of PCNA with yeast pol␩ is essential for its function in vivo [15]. An attractive model is that the stalled replication machinery can switch to different modes of lesion bypass through distinct PCNA modifications, since RAD5 and UBC13/MMS2 are specifically involved in error-free bypass of DNA damage by template switching to the allelic copy on the sister chromatid [11,16]. The finding that, apart from yeast, DNA damageinducible ubiquitination of PCNA occurs in human S-phase cells as well [14], indicates that this process is evolutionary conserved from yeast to mammals. Thus, the recruitment of TLS pol␩, ␫ and ␬ at PCNA foci in damaged mammalian S-phase cells (see later) might be directed by Rad6/Rad18-mediated ubiquitination of PCNA at stalled replication forks. Rad18-null mutant mouse- or chicken cells are (hyper)sensitive to various types of DNA damage and show a delay in DNA synthesis following UV exposure, indicating a defect in post-replication repair [17,18]. Similar results are found in human cells over-expressing dominant-negative Rad18, containing an amino acid change in the RING-finger motif [19]. In contrast to yeast, mutagenic TLS in vertebrate cells is only partly dependent on Rad18 activity

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since UV-induced mutagenesis is reduced but not abolished in Rad18-null mouse cells [18]. A possible candidate for Rad18-independent TLS could be pol␬, since defects in both Rad18 and pol␬ show an additive effect on UV-sensitivity [20], although a role of pol␬ in UV-induced mutagenesis in vivo needs to be established. To bypass persistent DNA lesions during S-phase, Rad18-null mutants rely on homologous recombination, as frequencies of spontaneously occurring- and DNA damage-induced sister chromatid exchanges (SCE) are increased compared to controls [17,18]. In chicken DT40 cells, this Rad18-independent hyper-recombination is fully mediated by Rad54-dependent homologous recombination, since Rad18−/− ; Rad54−/− double mutants are synthetic lethal [17]. Human and mouse Rad18 physically interact with both Rad6A and Rad6B, the two homologues of Rad6 in higher eukaryotes [19]. Analyses on the in vivo role of Rad6 on TLS in vertebrate cells is hampered due to the finding that simultaneous inactivation of both Rad6 homologues leads to non-viability [21,22]. Inactivation of Rad6B in mouse cells does not affect sensitivity to UV or ␥-rays [21], suggesting redundancy with Rad6A in recovery from DNA damage during S-phase. Rad6B-deficient mice show, in contrast to Rad6A-deficient mice, male infertility due to increased apoptosis of spermatocytes and impairment of elongating and condensing phases of spermatid development [21,22]. Interestingly, this phenotype is accompanied with an increase in meiotic crossing-overs [22] that possibly is caused by accumulation of double-strand DNA breaks. Apart from a putative role in the DNA damageinduced ubiquitination of PCNA, the human Rad6 homologues seem to be involved in a p53 response upon exposure of human cells to the topoisomerase II inhibitor adriamycin [23]. Human Rad6 homologues physically interact with p53 and, following induction of DNA damage, with other proteins of the p53 pathway. The Rad6 homologues ubiquitinate p53 and Rad6 proteins co-localise with ubiquitinated p53 in the nucleus of damaged cells, whereas in untreated cells human Rad6 is found in both nucleus and cytoplasm. The cellular response upon DNA damage results in alterations of the ubiquitination status of p53 and a cell cycle arrest at the G2 /Mphase.

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In keeping with the functional conservation of damage bypass mechanisms it has been shown that reduction of hMms2 expression in human cells, via antisense RNA expression, results in increased UV-induced mutagenesis [24]. This is associated with a reduction of error-free bypass of UV-induced DNA lesions using a mechanism involving gene conversion, probably by template switching. It is likely that elimination of this error-free damage avoidance pathway by disruption of hMms2 function probably channels DNA lesion bypass into the mutagenic TLS pathway [24]. Thus, very similar to the two ways of DNA damage bypass in yeast [16], vertebrates possess lesion bypass mechanisms that are error-free (mediated by template switching) and error-prone (mediated by TLS polymerases). It currently is not known what factors determine whether a particular DNA lesion is shuttled into either of the two pathways.

mation contains a C2H2 zinc-finger, a bipartite NLS and a PCNA-binding site [25]. Using site-directed mutagenesis, it has been shown that all three of these motifs are required for correct localisation of pol␩ in replication foci (Kannouche and Lehmann, personal communication). Pol␫ requires pol␩ for spot formation and co-localisation with PCNA, since in XP-V cells formation of pol␫ spots is found in only a minority of the cells after UV treatment and co-localisation with PCNA is abrogated [26]. Thus, pol␫, which has no nuclear localisation signal motif, is still localised in the nucleus in pol␩-deficient cells, suggesting that another protein than pol␩ is required for the nuclear import of pol␫. In addition to its heterodimerization with Rev3, mammalian Rev7 has been found to bind in vivo to Rev1 [27], supportive of the model that TLS in vivo is a multiprotein, multistage process.

4. Phenotypes of TLS polymerase mutants 3. Cell biology of TLS polymerases The effect of DNA damage on the intracellular distribution of TLS polymerases has been studied in human cells expressing pol␩, ␫ and ␬, tagged with Green Fluorescent Protein [13,25,26]. All three polymerases are homogeneously distributed throughout the nucleus, whereas 10–15% of the cells show nuclear spots. The latter frequency increases up to more than 60% when cells are exposed to replication-blocking agents such as UV, methyl methanesulphonate (MMS), hydroxyurea and cis-platin (CisPt), a cross-linking agent. Pol␩ and pol␫ co-localise with PCNA [26] and for pol␩ a co-localisation with bromodeoxyuridine (BrdU) spots is found [25], indicative for a role of these enzymes in DNA replication. Studies by Bergoglio et al. [13] suggest that the localisation of pol␬ is similar to that of pol␩ and pol␫. In contrast, recent work by Ogi et al. demonstrates that pol␬ co-localises with pol␩ in PCNA-containing replication factories in only a minority of the cells (Lehmann et al., personal communication). Pol␩ and its paralogue pol␫ show a physical interaction and are recruited to and co-localise at UV-damaged subnuclear sites in a highly co-ordinated fashion [25,26], suggesting the localisation of a complex of TLS polymerases at stalled replication forks at DNA lesions. The C-terminal 120 amino acids required for pol␩ spot for-

Amongst the polymerases involved in replicative bypass of DNA lesions, Rev1 and Rev3 play an important role in the viability of vertebrate cells. A Rev1 defect in chicken DT40 cells results in a reduced proliferation rate compared with wild type cells and an increased level of apoptosis that results in a poor clone forming ability [28], whereas Rev3 is essential for cellular and embryonic viability [29–33]. In addition, although normal replication and proliferation can occur in the absence of Rev3, Rev3-deficiency leads to increased apoptosis in embryos that is independent of p53 [31–33]. Both Rev1- and Rev3-deficient cells show elevated levels of spontaneous chromosomal aberrations indicative of the accumulation of double-strand breaks (DSB) [28,33]. These breaks may have resulted from collapsed replication forks at endogenous DNA lesions such as 8-oxo-guanine, abasic sites and 1-N6 -etheno-adenine which are substrates for the unique deoxycytidyl transferase activity of Rev1 in vitro [34]. The involvement of DNA damage in Rev1- and Rev3-dependent clastogenicity and toxicity is supported by the enhanced sensitivity of Rev3deficient blastocysts to N-acetoxy-acetylaminofluorene (NA-AAF), causing DNA lesions that are strong impediments for replication [33]. Increased sensitivity of Rev1-deficient chicken DT40 cells following

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treatments with UV, the UV-mimicking agent 4-NQO, hydrogen peroxide, X-rays and CisPt [28], suggests that Rev1, and probably also Rev3, plays a central (though not necessarily catalytic, see later) role in bypassing many different types of toxic DNA lesions in vivo. The finding that human cells expressing antisense RNAs against Rev1 or Rev3 show normal proliferation and no enhanced sensitivity for UV or (±)-7␤,8␣-dihydroxy-9␣,10␣-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) likely is caused by incomplete abolishment of Rev1- and Rev3-activity [35,36]. Probably, pol␩ and ␬ are required for bypass of a smaller spectrum of toxic DNA lesions as compared with Rev1. Endogenous DNA damage is efficiently bypassed in vertebrate cells defective for pol␩ or pol␬, since cell proliferation and viability are normal [20,37,38], and no increase of chromosomal aberrations is found for normal diploid XP-V cells and pol␬-deficient chicken DT40 cells [20,37]. In addition, human XP-V cells show only enhanced sensitivity to UV when pretreated with caffeine, a treatment that abolishes the S-phase checkpoint. Pol␬-deficient vertebrate cells show normal sensitivity to ␥- and X-rays, CisPt, and N-2-acetylaminofluorene (AAF) [20,38]. Both Pol␩ and ␬ show overlapping specificities in bypassing in vitro an abasic site, 8-oxo-guanine and dG-AAF and most of these lesions are also substrates for Rev1- and pol␫-mediated TLS (summarised in ref. [8]). However, pol␬ plays a significant role in bypassing lesions induced by the aryl hydrocarbon benzo(a)pyrene (B[a]P) and by UV in vivo. Mouse and chicken cells deficient for pol␬ are moderately sensitive for UV that is accompanied with higher frequencies of chromosomal aberrations [20,38,39], and a pol␬ defect in mouse embryonic stem (ES) cells results in increased sensitivity for B[a]P [38]. B[a]P-induced DNA lesions such as dG-N2 -BPDE are excellent substrates for pol␬-mediated TLS in vitro (summarised in ref. [8]). Normal cells are protected for B[a]P-induced toxicity, most likely via inducible expression of pol␬ that has two binding motifs in its promoter for a transcription factor that is activated upon binding of aromatic compounds such as B[a]P to the aryl hydrocarbon receptor [40]. In agreement, mice exposed to polyaromatic hydrocarbons show increased levels of pol␬ [40], which is expressed in epithelial cells of target tissues such as lung, stom-

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ach and skin as well as in cells of the adrenal cortex, a site of active steroid biosynthesis [41]. The UV-sensitivity of pol␬-deficient cells is more puzzling since cyclobutane pyrimidine dimers (CPD) and (6–4) pyrimidine–pyrimidones ((6–4) PP) are not bypassed by pol␬ in vitro (summarised in ref. [8]). Therefore, pol␬, like Rev1, might have a structural or accessory role in UV lesion bypass. SCE are often regarded as cytogenetic events that represent bypass of replication-blocking DNA lesions via a homologous recombination-mediated pathway. Interestingly, DNA damage-induced SCE and clastogenicity are differently affected upon inactivation of TLS polymerases in vertebrate cells. UV-induced chromosomal aberrations and SCE in diploid human cells are not dependent on pol␩ [37,42,43]. Possibly, due to inactivation of p53-dependent pathways and to defective S-phase checkpoint control, SCE are only enhanced when XP-V cells are immortalised or virally transformed [37,43]. In pol␬-defective chicken DT40 cells, both SCE and clastogenicity are increased after exposure to UV or 4-NQO [20]. However, in Rev1-defective chicken- or mouse-cells exposed to replication-blocking agents, the clastogenic response is enhanced, especially at a phase in late S or early G2 , whereas the induced levels of SCE remain normal ([28]; Jansen and De Wind, unpublished results). Thus, DSBs induced at persistent replication forks that are substrate for pol␩ or Rev1, are not shuttled in DSB repair, in contrast to breaks that occur in the absence of pol␬. Both pol␬ and Rev1 prevent SCE induced by replication blocks at endogenous DNA lesions, since spontaneous SCE is increased in chicken cells deficient for these proteins [20,28]. Although most TLS polymerases appear to co-localise, the analysis of cell cycle profiles of TLS polymerase-deficient cells provides additional clues as to their differential roles in DNA damage bypass during replication. In BrdU incorporation experiments, a delay during the entire S-phase is found in XP-V cells, compatible with the role of pol␩ in processing UV-induced DNA damage during all stages of the S-phase [43]. Conversely, pol␬ seems to act in early S-phase, since the UV-sensitivity in pol␬-deficient chicken DT40 cells specifically is increased for cells that are in early S-phase [20]. Untreated Rev1-deficient DT40 cell populations show a reduction of S-phase cells accompanied with an

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increase of G2 /M-phase cells and of apoptotic cells compared with wild type cells. CisPt treatment accentuates this phenotype [28]. In addition, mouse embryonic stem cells containing a deletion of the N-terminal BRCT domain of Rev1 (Rev1BRCT/BRCT ES cells) show a transient delay at late S- and at G2 /M-phases following UV exposure. Of note, unlike XP-V cells, Rev1BRCT/BRCT ES cells are not sensitised by caffeine after UV exposure (Jansen and De Wind, unpublished results), supporting the differential functions of both proteins in UV-induced DNA damage bypass.

5. TLS polymerases and DNA damage-induced mutagenesis Vertebrate homologous of Rev1 and Rev3 probably play a central role in DNA damage-induced mutagenesis in vivo. Mouse Rev1BRCT/BRCT ES cells as well as diploid human cells expressing antisense mRNAs for hRev1 or hRev3 show a reduction of UV-induced mutations of 64–94%, depending on the cell line tested ([35,36], Jansen and de Wind, unpublished results). Similar results were found for BPDE-exposed human cells [36]. It is not clear whether the difference in the mutability of antisense expressing cell lines is due to differences in Rev1 or Rev3 protein levels, as these proteins are hard to detect by immunoblot analysis [35,36]. Although UV mutation spectra have not been reported, the spectrum of BPDE-induced mutations in cells expressing antisense hRev3 mRNA is similar to that of control cells [36], suggesting that Rev3 plays a role in bypassing all types of BPDE-induced DNA lesions. Concerning UV damage, Rev3 efficiently extends in vitro ‘mismatches’ introduced by another TLS polymerase opposite the 3 pyrimidine of the lesion [44–46]. Lack of Rev3 may lead to an accumulation of permanently arrested replication forks stalled at lesions, resulting in apoptosis. The role of Rev1 in UV mutagenesis is poorly understood, since (i) mutational spectra indicate that base pair changes are not caused by insertion of deoxycytosine opposite the UV lesion [47,48], and (ii) both CPD and (6–4) PP are poor substrates for TLS in vitro by Rev1 (reviewed in refs. [7,8]). However, instead of using its catalytic activity, Rev1 could direct appropriate TLS polymerases for efficient bypass of UV lesions in ver-

tebrate cells, explaining the important role of Rev1 in DNA damage-induced mutagenesis. Human XP-V cells are hypermutable by UV to the same extent as NER-deficient cells [49,50], suggesting that pol␩ protects human cells for UV-induced mutagenesis. It is well established that pol␩ efficiently performs error-free TLS on CPD (summarised in ref. [8]). Although pol␫ is the only known TLS polymerase besides pol␩ that is capable of inserting nucleotides opposite CPD and (6–4) PP in vitro (reviewed in ref. [9]), the apparent lack of functionality of pol␫ in XP-V cells [26] suggests that this polymerase is not causing the hypermutable phenotype of XP-V cells. The error-prone TLS activity in XP-V cells is characterised by inserting Ts opposite UV lesions, since GC to TA as well as AT to TA transversions dominate the spectrum of UV-induced hprt gene mutations in XP-V cells, whereas transitions at GC base pairs dominate spectra in normal or NER-deficient cells [48]. The lack of pol␩ in XP-V cells also affects the strand specificity of the mutations [48]. UV exposure of normal G1 -phase cells results in transitions at GC base pairs that are mainly caused by lesions in the non-transcribed strand, whereas normal cells irradiated in S-phase show mutations due to lesions predominantly in the transcribed strand. UV-exposed XP-V cells show a lack of strand specificity irrespective the cell cycle phase [48]. These results suggest that pol␩ uses predominantly the non-transcribed strand as template for TLS and may indicate a bias for TLS polymerases in their role in leading strand or lagging strand DNA synthesis. Pol␬ performs error-free bypass in vitro of dG-N2 -BPDE lesions (summarised in ref. [8]) and protects vertebrate cells for the mutagenic effects of dG-N2 -BPDE, since B[a]P-induced hprt mutagenesis in pol␬-deficient ES cells is about 10-fold higher than in wild type cells [38]. B[a]P induces predominantly mutations at GC base pairs and mutational spectra analysis shows mostly base pair changes at GC base pairs accompanied with a strong shift towards GC to TA transversions in pol␬-deficient cells as compared to control cells [38]. Most of the mutations are probably caused by adducts in the non-transcribed strand in both genotypes [38]. In pol␬-deficient cells, pol␩ activity is probably responsible for most TLS past BPDE-induced DNA adducts due to preferential incorporation of A (and to a lesser extent G or C)

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opposite dG-N2 -BPDE adducts as shown in in vitro assays (summarised in ref. [8]). Ectopic expression of pol␬ in human and mouse cells increases spontaneous mutagenesis 9–16-fold [12,13]. In the mouse study, approximately 30% of these mutants contain frameshift mutations at single base pairs [12]. Of note, pol␬ is over-expressed in some lung tumours, supporting a role of pol␬-mediated TLS in tobacco smoke-induced mutagenesis that underlies the development of lung cancer [51].

6. Involvement of TLS polymerases in somatic hypermutation Somatic hypermutation (SHM) is a highly mutagenic process that occurs in antigen-stimulated B cells in the germinal centres of the immune system. SHM is required to enable the generation of antibodies of high affinity, needed to defend vertebrates against life-threatening antigens [52,53]. SHM introduces almost exclusively base pair alterations and is restricted to a 1–2 kb pair genomic region encoding the variable, antigen-binding, section of an immunoglobulin (Ig) protein. The mutation rate in hypermutating B cells is an astonishing 10−3 per base pair per generation, which is about one million-fold higher than in normal somatic cells. This clearly suggests the involvement of an error-prone replication or repair process. SMH affects both A-T and G-C base pairs, although the frequencies of mutations at A-T and G-C base pairs may differ markedly between different model systems. For example, in mouse and human hypermutated B cells the frequency of mutations at A-T base pairs is close to 50%, whereas the human BL2 lymphoma cells show only 20% of the mutations at A-Ts. Furthermore, many B cells contain multiple mutations per variable gene following SHM. SHM is a rapid process as shown by a study reporting the occurrence of misincorporations already 90 min after induction of SHM in vitro in a human B cell line [54]. Since their identification, TLS polymerases are excellent candidates to participate in SHM due to their lack of 3 –5 proofreading activity and, consequently, their low fidelity in replicating DNA, with mutation rates in vitro up to 10−1 per base pair [10]. Moreover, SMH most likely depends on replication of a damaged DNA template that requires the activity of

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TLS polymerases. Such DNA damage can be induced by activation-induced deaminase (AID), an enzyme that is exclusively expressed in antigen-stimulated B cells in the germinal centres and which activity is essential for SHM in chicken-, mouse- and human cells [53,55]. AID deaminates cytosine, to uracil, in single-stranded DNA, but not in RNA or in double-stranded DNA [56–58]. Removal of uracil from the DNA by uracil-DNA glycosylases (UDG) results in the formation of abasic sites that are excellent substrates for pol␫, pol␬ and Rev1 in vitro, and to a lesser extent for pol␩ (summarised in ref. [8]). Abasic sites are, by definition, non-instructive DNA lesions and therefore highly mutagenic. Also uracil residues could be involved in SHM, since these residues will be recognised as thymine by the replication machinery resulting in GC to AT transitions in the subsequent round of replication. Indeed, SHM is slightly increased in B cells of UDG-deficient Ung−/− mice as well as in chicken DT40 cells where UDG activity is inhibited [59,60] and mutational spectra analyses shows that this increase is accompanied with an elevated frequency of GC to AT transitions. It should be noted, however, that SMH is not restricted to G-C base pairs [52,53,61]. This strongly suggests that the generation of uracils in DNA by AID is just an initiating event for SHM and that error-prone repair or bypass replication beyond these residues results in mutations found at both A-T and G-C base pairs. Which TLS polymerases may be involved in SHM and what could be their role in this process? Evidence exists that both pol␨ and Rev1 play an important role in SHM [28,62,63]. Rev3 is upregulated in germinal centre cells as well as in a human lymphoma cell line undergoing SHM [63]. Mice expressing antisense Rev3 mRNA show a reduced number of B cells in lymphoid tissues, whereas germinal centres are abundantly present and large in size [62]. This suggests that normal replication of B cells is not affected. More importantly, antisense Rev3 mice show a 43% lower mutation frequency in hypermutated B cells compared with controls [62]. Comparable results are found in a human B cell line treated with an antisense oligonucleotide against Rev3 [63]. Mutational spectra analysis shows no difference between cells with reduced expression of Rev3 and normal levels of Rev3, indicating that SHM at both A-Tand G-C-base pairs is affected by reduction of pol␨.

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Possibly, reduction of Rev3 expression interferes with efficient extension of mismatches introduced by other DNA polymerases, which results in aberrant replication intermediates signalling to apoptosis. An alternative role of Rev3 in SHM is suggested by two independent findings. Firstly, double-strand DNA breaks are frequently found at mutational hotspots in cells undergoing SHM (reviewed in [52]) (although the function of these breaks in the process of SHM is still unclear). Secondly, Rev3 is responsible for the induction of mutations in DNA sequences that occur during homology-mediated repair of a double-strand DNA break in yeast [64,65]. Thus, Rev3 might induce SHM at double-strand DNA breaks that are associated with variable Ig gene regions undergoing SHM. The role of Rev1 in SHM has recently been studied in chicken DT40 cells that hypermutate variable regions of Ig genes mostly via gene conversion events using upstream pseudogenes as donors. Nevertheless, one-third of the SHM events in DT40 cells is caused by error-prone DNA synthesis [28]. The latter type of SHM is markedly diminished in chicken DT40 cells containing a disruption of the Rev1 gene, resulting in a mutation frequency that is close to the calculated methodological background error [28]. At a first glance, this result is rather surprising, since the only known catalytic activity of Rev1 is a DNA-dependent deoxycytidyl transferase activity opposite predominantly guanine, uracil and abasic sites ([34], reviewed in [8]). However, it is possible that mismatches induced by Rev1 at uracil and abasic sites are preferentially extended by pol␨ thereby introducing misincorporations downstream of these mismatches that, in addition to alterations at G-C base pairs, also may lead to changes at A-T base pairs. Alternatively, Rev1 might be a key player in assembling and regulating a putative mutasome that includes different TLS polymerases responsible for SHM. Rev1 KO cells may lack such a mutasome resulting in a reduction of mutations. In the human lymphoma cell line BL2, induction of SHM activity is correlated with a decrease in expression of Rev1 mRNA [66]. Studies on rearranged variable heavy chain Ig genes of XP-V patients suggest that pol␩ plays only a minor role in SHM as compared with Rev1 or pol␨ [67,68]. XP-V patients show a normal level of SHM, i.e. the proportion of mutated clones as well as the mutation frequency is similar to those of controls. However,

XP-V patients show a bias towards G-C base pairs accompanied with a decrease of about 60% in the proportion of alterations at A-T base pairs, in particular at AA or TA motifs in the non-transcribed strand according to one study [67]. Thus, pol␩ appears to act in a strand specific manner in the generation of mutations at A-T base pairs during SHM. This hypothesis is supported by comparisons of mutational spectra analysis of Ig genes and mutations induced by pol␩ following replication in vitro of a bacterial gene or a mouse variable region Ig gene [67,69,70]. Somatic hypermutation results in the formation of productive- and non-productive rearrangements of Ig genes of which the latter represents an early stage of SHM prior to selection of high affinity antibody secreting B cells. A comparison of mutational spectra of both types of rearrangements in Ig genes of XP-V patients reveals the presence of small deletions and insertions in the non-productive rearrangements only [68]. These alterations could be attributed to the action of other mutagenic polymerases than pol␩, such as pol␬. However, pol␬-deficient mice show a normal immune response upon challenging with an antigen and germinal centre B cells mutated their Ig genes normally, suggesting that pol␬ is not involved in SHM [39,71]. The physical interaction of pol␩ with pol␫ as described above alludes to a role for pol␫ in SHM. In accordance, expression of pol␫ is upregulated during SHM in human cells [63,66]. Indeed, the human Burkitt’s lymphoma cell line BL2 deficient for pol␫ shows complete loss of SHM [72]. These results, backed by biochemical properties of pol␫, suggest that this polymerase may insert efficiently an adenosine nucleotide opposite an AID-induced uracil or abasic site. Alternatively, its 5 -deoxyribose phosphate lyase activity (reviewed in ref. [9]) suggests that pol␫ might be involved in long patch base excision repair of G-U mismatches. However, recent studies with 129/SvJ mice that contain a premature translational stop codon in a 5 -exon of the endogenous Rad30B gene, encoding Polymerase ␫ suggest that pol␫-deficient mice undergo essentially normal SHM [73]. It should be mentioned, though, that the spectra of mutations in hypermutating normal BL2 cells and other lymphoma cell lines, which are characterised by a strong bias towards mutations at G-C base pairs, is much different to that of normal human B cells [52,61,72]. Also AID expression is not induced following stimulation

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