DNA POLYMERASES h AND i

By ALEXANDRA VAISMAN,* ALAN R. LEHMANN, � AND ROGER WOODGATE*

*Laboratory of Genomic Integrity, National Institute of Child Health and Human Development,

National Institutes of Health, Bethesda, Maryland, 20892-2725�Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton BN1 9RQ,

United Kingdom

I. Historical Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205II. Identification of RAD30 and its Orthologs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206

III. Biochemical Properties of Pol� and Pol� . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207IV. Translesion Synthesis by Pol� and Pol� . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210V. Structure of the Catalytic Core of S. Cerevisiae Pol� . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212

VI. Regulation and Localization of Pol� and Pol�. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215VII. Mutations in Pol� in XP Variants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217

VIII. Pols � and � and the Polymerase Switch: Interactions with PCNA and Rev1 . . . 219IX. Protection from Cellular Effects of DNA Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 220X. Roles of Pol� and Pol� in Somatic Hypermutation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

I. HISTORICAL PERSPECTIVE

Based on genetic studies in Escherichia coli, it was believed for many yearsthat damage-induced mutations in DNA were generated in a two-stepprocess commonly referred to as Translesion DNA Synthesis (TLS) (fora general review, see Friedberg et al., 1995). The first step of this process is(mis)incorporation and involves the physical insertion of a nucleotideopposite the DNA lesion. The second step involves extension of the(mis)incorporated base, such that the replication-blocking lesion iscompletely bypassed. It was originally hypothesized that both steps wereperformed by the cell’s high-fidelity replicase, Pol III, with the assistance ofaccessory proteins, such as RecA and UmuDC, which together facilitatedhighly error-prone DNA replication through various DNA lesions (Bridgesand Woodgate, 1984, 1985a,b).

The first clue that cells may actually contain specific polymerasesspecialized in lesion bypass came in 1996 with the purification of Pol�, aheterodimer consisting of Rev3/Rev7, which has the ability to replicate pastultraviolet (UV) induced thymine–thymine cis–syn cyclobutane dimers(CPDs) (Nelson et al., 1996b) (reviewed in detail in Chapter 6). At thesame time, it was found that the product of the Saccharomyces cerevisiae REV1gene long implicated in the mutagenic process has deoxycytidyl transferaseactivity (Nelson et al., 1996a). Although the REV3 gene encoding thecatalytic subdomain of Pol� belongs to the B-family of DNA polymerases,

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the REV1 gene exhibits no sequence or motif homology to any of thepreviously identified DNA polymerase families. Instead, it shares homologywith the umuC gene (Larimer et al., 1989) that is required for damage-induced mutagenesis in E. coli (Kato and Shinoura, 1977; Steinborn, 1978).Because at that time Rev1 was believed to use only dCTP as a substrate, thepossibility that UmuC or phylogenetically related proteins might actuallypossess DNA polymerase activity was largely overlooked. However, that viewbegan to change in the late 1990s, when it was shown that a highly purifiedpreparation of heterodimeric E. coli UmuD0

2C was able to bypass a syntheticabasic site unassisted (Tang et al., 1998). Although at that time there was apossibility that the UmuD0

2C preparation contained a trace amount ofendogenous PolIII, the biochemical studies on UmuD0

2C provided the firsthint that many of the proteins implicated in the mutagenic process mightactually be bona fide DNA polymerases. The intrinsic polymerase activity ofthe UmuD0

2C complex was subsequently confirmed (Tang et al., 1999) andwas shown to reside in the UmuC subunit of the complex (Reuven et al.,1999). Amazingly, within a short, �18-month period, DNA polymeraseactivity was also described for several proteins homologous to UmuC andRev1. These include E. coli PolIV, encoded by the dinB gene (Wagner et al.,1999) and described in detail in Chapter 8; its human homolog Pol�( Johnson et al., 2000a; Ohashi et al., 2000; Zhang et al., 2000c), describedin Chapter 9; and two eukaryotic paralogs of S. cerevisiae RAD30, Pol�( Johnson et al., 1999b; Masutani et al., 1999) and Pol� ( Johnson et al.,2000b; Tissier et al., 2000b; Zhang et al., 2000b), which are described indetail later. Collectively, these proteins are now referred to as the Y-family ofDNA polymerases (Ohmori et al., 2001).

Over 200 proteins that share homology to the Y-family polymerases havesince been identified in prokaryotes, archaea, and eukayrotes. Interesting-ly, many organisms often possess more than one family member, suggest-ing that Y-family polymerases play important roles in cellular survival orevolutionary ‘‘fitness’’ (Friedberg et al., 2002; Yeiser et al., 2002). Indeed,defects in human Pol� result in the sunlight-sensitive and cancer-proneXeroderma pigmentosum variant (XP-V) syndrome ( Johnson et al., 1999a;Masutani et al., 1999) (see following), whereas mutations in E. coli dinBreduces the cell’s ability to undergo adaptive mutagenesis in stationaryphase (McKenzie et al., 2001; Tompkins et al., 2003).

II. IDENTIFICATION OF RAD30 AND ITS ORTHOLOGS

The RAD30 gene was identified in a search of the S. cerevisiae genome forhomologs of prokaryotic DinB- and UmuC-like proteins and for eukaryoticRevl-like proteins (McDonald et al., 1997; Roush et al., 1998). Similar to its

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E. coli counterparts, RAD30 is damage inducible, and Rad30-disruptedyeast strains are mildly sensitive to UV light (McDonald et al., 1997; Roushet al., 1998). Epistasis analysis suggested an involvement of the Rad30protein in the postreplication repair pathway (McDonald et al., 1997),and an important step in understanding the cellular function of Rad30came with its overproduction and purification ( Johnson et al., 1999b).Biochemical characterization revealed that the Rad30 protein is a bona fideDNA polymerase, which uses all four nucleoside triphosphates in a tem-plate-dependent reaction. Strikingly, however, Rad30 was shown to bypassa thymine–thymine CPD exceptionally efficiently and relatively accurately( Johnson et al., 1999b). As the sixth eukaryotic polymerase reported in theliterature at that time, the S. cerevisiae Rad30 protein was designatedas Pol�.

Shortly afterward, a human homolog of RAD30 was identified andshown to be the enzyme mutated or missing in cells from humans exhibit-ing the XP-V phenotype ( Johnson et al., 1999a; Masutani et al., 1999). XP-Vcells are proficient in nucleotide excision repair but defective in postre-plicative repair (they have a reduced ability to make intact daughter DNAstrands on damaged templates) (Lehmann et al., 1975). This defect resultsin an increase in UV-induced mutagenesis and leads to a high incidenceof skin cancer (Cleaver and Carter, 1973). The name approved by theHuman Genome Organization for the human RAD30 ortholog is POLH,although it is also commonly referred to as the XPV or RAD30A gene.

Soon after the discovery of human POLH, a second human homolog ofRAD30 was identified and initially designated RAD30B (McDonald et al.,1999). Subsequent studies demonstrated that, similar to the other phylo-genetically related proteins, the Rad30B protein has DNA polymeraseactivity. The protein was therefore renamed DNA polymerase � and isencoded by the human POLI gene (Tissier et al., 2000b).

POLH orthologs are evolutionally conserved in a wide variety of eukar-yotes from yeast to human, and it is thought that POLI most likely arose asa gene duplication of POLH during evolution of the species. POLI ’sdistribution is much more limited than that of POLH, and it is foundmostly in higher eukaryotes.

III. BIOCHEMICAL PROPERTIES OF POL� AND POL�

Similar to other members of the Y-family, both Pol� and Pol� are highlyerror prone and lack intrinsic exonuclease activity, meaning they cannotproofread any of the errors they make when copying DNA. Both enzymesare distributive and incorporate only a few deoxynucleotides beforedissociating from the extended product. There are, however, significant

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differences in the biochemical properties of these phylogenetically relatedenzymes. For example, a property that distinguishes human Pol� from Pol�and other members of Y-family DNA polymerases is its intrinsic 50-deoxyri-bose phosphate (dRP) lyase activity (Bebenek et al., 2001b; Prasad et al.,2003); that is, the ability to catalyze excision of 50-dRP from DNA duringbase-excision repair. Furthermore, Pol� has stronger dRP lyase activityrelative to its polymerase activity than does the main BER polymerase,Pol� (Prasad et al., 2003).

The fidelity of both yeast and human Pol� is very low, with in vitromisincorporation frequencies ranging from 10�2 to 10�3 (Fig. 1A; Johnsonet al., 2000c; Matsuda et al., 2000; Washington et al., 1999), which is at least100 times less accurate than that of replicative DNA polymerases (Kunkel,2004). As is usually seen with other DNA polymerases, error rates varydepending on mismatch composition and template sequence context(Goodman and Fygenson, 1998; Goodman et al., 1993; Mendelman et al.,1989; Zhang and Mathews, 1995). For example, higher misincorporationrates are observed when the base-pair 50 of the error is T:A or A:Tcompared to C:G or G:C (Matsuda et al., 2000). Pol�’s misincorporationfrequency and specificity is unprecedented, even when compared to Pol�.In general, Pol� forms most mispairs more frequently than Pol� (Fig. 1A).Furthermore, Pol� is characterized by a significantly broader range ofmisincorporation frequencies for different mispairs compared to Pol�(Fig. 1, compare distribution of points along x - and y -axes).

The fidelity of Pol� is uniquely template dependent. On primed single-stranded DNA, the most accurate nucleotide incorporation is observedopposite template A (with a misinsertion frequency of about 1 � 10�4)(Tissier et al., 2000b). The preference for the correct nucleotide in thiscase is ensured by tighter binding and faster incorporation compared tothe binding and incorporation of the incorrect nucleotide (Washingtonet al., 2004). In contrast, Pol� makes an extremely high level of errors attemplate T. In fact, depending on the sequence context, the wobble base,G, is incorporated 3 to 11 times more often opposite T than the Watson–Crick base, A ( Johnson et al., 2000b; Tissier et al., 2000b; Vaisman et al.,2001). On DNA templates with a 1-nucleotide 50 overhang, the pattern ofnucleotide misincorporation by Pol� is entirely different from that onprimed single-stranded DNA templates. In this case, Pol� is most inaccu-rate on template C, where C and A are misinserted three- to eight-foldmore often than the correct base, G (Frank et al., 2001).

Pol� and Pol� are characterized not only by high levels of nucleotidemisinsertion but also by their relatively high efficiency of mismatch exten-sion compared to classical DNA polymerases. The range of single mismatchextension frequencies by Pol� and both yeast and human Pol� are similar

FIG. 1. Comparison of insertion and extension fidelities of human Pols � and �.(A) Efficiencies of misincorporation ( finc) by Pol� (Tissier et al., 2000b) were plottedversus efficiencies of misincorporation by Pol� ( Johnson et al., 2000c; Washington et al.,1999), which is at least 100 times less accurate than that of replicative DNA polymerases(Kunkel, 2004 #1346; Washington et al., 1999). The dashed line corresponds tofinc Pol� ¼ finc Pol�. Points above the dashed line correspond to a higher frequency ofnucleotide misinsertion catalyzed by Pol� compared with that of Pol�, whereas pointsbelow the dashed line represent the reverse situation. In the base mispairs shown, thefirst base is the incoming nucleotide and the second base is in the template. (B)Efficiencies of mispair extension ( f 0

ext) by Pol� (Vaisman et al., 2001) were plotted versusthe efficiencies of mispair extension by Pol� (Washington et al., 2001). The dashed linecorresponds to f 0

ext Pol� ¼ f 0ext Pol�. Points above the dashed line correspond to a higher

relative efficiency of mispair extension catalyzed by Pol� compared with that of Pol�,whereas points below the dashed line represent the reverse situation. In the basemispairs shown, the first base at the 30primer terminus and the second base is in thetemplate.

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(Fig. 1B; Vaisman et al., 2001; Washington et al., 2001). Mismatch extensionby human Pol� is somewhat less efficient than the incorporation of thewrong nucleotide for most mispairs (Fig. 1; Washington et al., 2001). ForPol�, the correlation between relative efficiencies of mismatch formationand extension is more dependent on the identity of a mispair and the nexttemplate nucleotide encountered (Fig. 1; Vaisman et al., 2001). More mis-pairs are extended by Pol�, with a higher efficiency than by Pol� (Fig. 1B;Matsuda et al., 2000; Vaisman et al., 2001; Washington et al., 2001). AlthoughPol� is able to extend a variety of mismatches quite efficiently, a ‘‘buried’’mispair located 2–3 bases from the nascent primer chain significantlyinhibits further primer elongation and therefore restricts Pol� synthesisto short regions of DNA (Vaisman et al., 2001). Pol� is also essentially

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blocked by tandem mispairs, whereas human Pol� extends tandem double-mismatched termini at rates similar to those at which other polymerasesextend single mismatches (Matsuda et al., 2000). Human Pol� also generatesa remarkable variety of nucleotide deletion and addition errors with highfrequencies (Matsuda et al., 2000). These frameshift errors are explained bystrand slippage, often preceded by nucleotide misincorporation.

IV. TRANSLESION SYNTHESIS BY POL� AND POL�

The ability of Pol� and Pol� to bypass a variety of structurally diverseand potentially mutagenic lesions in vitro has been analyzed extensively.These biochemical studies revealed that the efficiency and patternof nucleotide incorporation opposite DNA damaged sites is distinct forthe two Rad30 paralogs. In some cases, Pol� readily bypasses a lesion,whereas Pol� does it inefficiently, or not at all. An example of Pol�’sand Pol�’s disparate TLS activities is observed at thymine CPDs andcisplatin-induced dG intrastrand crosslinks. Pol� efficiently facilitates ro-bust bypass of both CPDs ( Johnson et al., 2000c; Masutani et al., 2000;McCulloch et al., 2004) and platinum-GG cross links (Vaisman et al., 2000)in vitro, whereas Pol� bypasses the CPD in a limited manner (Tissier et al.,2000a) that is sequence context dependent (Vaisman et al., 2003) andis incapable of nucleotide incorporation opposite a cisplatin adduct(McDonald et al., 2001).

On the contrary, Pol� appears to be limited to insertion opposite the30 T of UV-induced 6-4 pyrimidine-pyrimidone dimers (6-4PP) and issignificantly inhibited after nucleotide incorporation at abasic sites andN-2-acetylaminofluorene (AAF)-modified guanine adducts (Haracska et al.,2001c; Kusumoto et al., 2002; Zhang et al., 2000a), whereas Pol� is signifi-cantly more efficient at incorporating across from both Ts of the 6-4PP(Tissier et al., 2000a; Vaisman et al., 2003) and opposite an abasic site( Johnson et al., 2000b; McDonald et al., 2001). When encountering certainother lesions, the enzymes behave in a similar manner. For example,both Pols can readily bypass an 8-oxoG lesion (Vaisman and Woodgate,2001; Zhang et al., 2000a), and both are limited to inefficient incor-poration opposite acrolein-derived deoxyguanosine (Minko et al., 2003;Yang et al., 2003) and benzo[a]pyrene (BP) adducts (Chiapperino et al.,2002; Frank et al., 2002; Rechkoblit et al., 2002). Thus, TLS not onlydepends on the particular lesion encountered but also on the polymeraseattempting TLS.

The accuracy with which any given lesion is bypassed is also uniquelydependent on each polymerase. The fact that humans lacking Pol� havean increased incidence of skin cancers clearly indicates that Pol� plays an

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important role in the accurate postreplication repair of UV-inducedCPDs in vivo. Depending on the point of view taken, the fidelity of bypassof CPDs in vitro by Pol� can be regarded as error prone or relativelyerror free. Using a template containing a CPD, ‘‘incorrect bases’’ oppositethe Ts are inserted at a frequency of approximately 10�2 ( Johnson et al.,2000c; Masutani et al., 2000; McCulloch et al., 2004), which could beregarded as error prone. However, this means that in 99% of inst-ances, Pol� inserts the ‘‘correct’’ bases opposite these lesions. No otherpolymerase displays any comparable accuracy.

With other lesions, Pol� frequently misinserts erroneous bases. Forexample, dG is predominantly misinserted at the 30T of the 6-4PP beforeDNA synthesis is aborted ( Johnson et al., 2001; Masutani et al., 2000). Pol�also predominantly misincorporates dA, and to a lesser degree dG and dT,at acrolein-derived dG (Yang et al., 2003), dA at cyclodeoxyadenosine(Kuraoka et al., 2001), dT at O6-methylguanine (Haracska et al., 2002),and dG and dA opposite BP-G adducts (Chiapperino et al., 2002). On thebasis of highly error-prone nature of the enzyme at these specific lesionsin vitro, one has to speculate that either these errors are removed in vivo byexonucleolytic proofreading (Bebenek et al., 2001a) or TLS of the lesionsis facilitated by a more accurate enzyme.

The specificity of nucleotide misincorporation opposite many of thelesions tested is very different for Pol� compared to that of Pol�. Pol� ischaracterized by unique nucleotide selectivity for most of the damagedsites and often incorporates the wrong nucleotide more efficiently thanthe correct one. Despite the fact that coding properties of damaged basesessentially depend on chemical structure, the miscoding potential of DNAlesions for Pol� appears to be related to its fidelity on undamaged DNA.The selection of nucleotides by Pol� is most unpredictable at template T.Depending on the surrounding sequence context and the type of damage,A, T, or G is predominantly inserted. An unusually accurate incorporationis observed at the 30T of 6-4PP in an ATTC sequence context (Vaismanet al., 2003). This exception is particularly interesting because structuralfeatures of 6-4PP-containing duplex DNA suggest that 30T of 6-4PP forms amore stable base pair with G than with A (Kim and Choi, 1995; Lee et al.,1999), which is consistent with the misincorporation specificity of otherDNA polymerases, including Pol� ( Johnson et al., 2001).

In contrast to the extremely promiscuous behavior of Pol� on templateT, the enzyme is very accurate at template A, which is either undamaged orcontains various stereoisomers of BP adducts placed in different sequencecontexts (Frank et al., 2002). Although the misincorporation frequencyincreases upon formation of BP adducts at adenosine, the fidelity of Pol�remains high (Frank et al., 2002). Thus, it is possible that while Pol� has

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evolved to protect us from damage occurring at thymine, Pol� may simi-larly help protect us from mutations at damaged adenosines.

In addition to yeast and human Pol� and human Pol�, Drosophila andmouse Pol� and Pol� enzymes have been overproduced and characterized(Ishikawa et al., 2001; Matsuda et al., 2001; McDonald et al., 2003).Although the enzymatic properties of the various Pol�s were similar fromall sources, the properties of Drosophila Pol� were very different from eitherthe human or mouse Pol� enzymes. In fact, Drosophila Pol� is more akin toPol� in its ability to traverse a CPD accurately (Ishikawa et al., 2001). Suchobservations lend support to the notion that Pol� arose as a duplication ofPol� and that evolutionary selective pressures acted on Pol� over themillennia, so as to distinguish it from Pol�.

V. STRUCTURE OF THE CATALYTIC CORE OF S. CEREVISIAE POL�

Although human Pols � and � are only �23% identical to each other atthe primary sequence level, they nevertheless have conserved blocks ofamino acids (called motif I–V in Kulaeva et al., 1996), which are readilyidentifiable when their primary amino acid sequences are aligned to otherY-family polymerases (Fig. 2A). These five motifs are located in theN-terminal half of the two proteins, which is now known to contain thecatalytic active site of all Y-family polymerases (Ling et al., 2001; Silvian et al.,2001; Trincao et al., 2001; Zhou et al., 2001). Remarkably, even thoughthere is little to no similarity in the primary amino acid sequence of Y-family polymerases with those previously identified from the A-, B-, C-, D-,or X-polymerase families, the three-dimensional structure of Pol� is similarto those previously reported for other DNA polymerases, in that it retainsthe overall topology of a right hand that is capable of holding DNA in itsgrasp (Fig 2B). Similar to the classical polymerases, the catalytic core ofPol� is composed of a thumb subdomain, which is thought to have aprimary role in DNA duplex binding and polymerase processivity; a fingerssubdomain, which is likely to be important for nucleoside triphosphate(dNTP) selection; and a palm subdomain, which contains the catalyticallyimportant residues for the nucleotidyl transfer reaction.

The core of the palm domain of Pol� includes a four-stranded antipar-allel � sheet flanked by two small � helices and an �-helical structuresituated at the base of the palm, which is superimposable with the core ofthe palm domain from high-fidelity polymerases. Akin to the active sitesof the polymerases from the A-, B-, C-, and X-families, the palm domain ofPol� contains three carboxylates (Asp30 and Asp155-Glu156), which coor-dinate two catalytically essential metal ions assisting the nucleotidyl trans-ferase reaction. Although the palm subdomain is nearly superimposable

FIG. 2. Cartoon representation of the conserved regions in Pols � and � and thecrystal structure of the catalytic core of Saccharomyces cerevisiae Pol�. (A): Schematicalignment of hPOLH and hPOLI. Conserved amino acid sequences found in all Y-familypolymerases are represented by the five colored boxes (motifs I–V) and by white boxesindicating unique sequences. Gaps have been introduced in the sequences for optimalalignment. The length of Pols � and � are indicated by the number of amino acids onthe right site of the diagram. A conserved zinc-binding motif in the C terminus of Pol�is indicated as C2H2 in the gray box. Structural domains of Pol� shown in blue (finger,F), red (palm, P), green (thumb, T), and purple (little finger, LF) are indicated abovethe alignment. The positions of the nuclear localization signal (NLS), the domaininvolved in polymerase localizing into replication foci (FS), and regions responsible forthe interaction with other proteins are also indicated. (B) Structural domains of Pol�are shown in red (palm), green (thumb), blue (finger), and purple (little finger) as aribbon diagram. The acidic residues (Asp30, Asp155, and Glu156) that make up theactive site are shown as gold rods. S. cerevisiae Pol� has an insert of �70 amino acids inthe palm domain that is absent in the archaeal Y-family polymerases (Ling et al., 2001;Silvian et al., 2001; Zhou et al., 2001), and this region is shown in pink. This figure wasmade with ribbons (Carson, 1987).

DNA POLYMERASES � AND � 213

214 VAISMAN ET AL.

on the palm of high-fidelity polymerases, the fingers and the thumbdomains of Pol� are small and stubby when compared to the fingersand thumb of polymerases from other families. These structural featuresprovide a basis for the tolerance by Pol� for mismatch- or lesion-inducedgeometric distortions in the DNA. The fingers subdomain in Pol� consistsof a �-sheet and three small � helices (Fig. 2). The ‘‘O helices,’’ which arebelieved to play a key role in fidelity enhancement by tightening theinterface between the polymerase and the replicating base pair, are absentin the fingers of Pol�. Instead, only a short loop is positioned to potentiallyinteract with the replicating base pair. The thumb subdomain of Pol� iscomposed of six � helices that are structurally distinct from helices inother DNA polymerases (Fig. 2B).

The surface area in the Pol� active site enclosed by the palm, fingers,and thumb domains is reduced compared to classical polymerasesand appears to be too small to allow for efficient binding and replicatingDNA. To increase its binding area, Pol� employs an additional C-terminaldomain, which mimics an extra set of fingers and has been termedthe polymerase-associated domain (PAD) (Trincao et al., 2001). Thisdomain is not found in DNA polymerases from other families butis structurally conserved within the Y-family polymerases. In archaeal DinBhomologs Dpo4 and Dbh, it has been termed the ‘‘little finger’’ (Ling et al.,2001) and the ‘‘wrist,’’ respectively (Silvian et al., 2001), and we will referto this domain as the little finger (LF) throughout the rest of this chapter.The LF domain has been proposed to play a role similar to that of theaccessory factors employed by other polymerases, so as to increase theprocessivity of the polymerase (Boudsocq et al., 2004). This domain in Pol�consists of an antiparallel � sheet and two long � helices packed next tothe fingers subdomain and is connected to the thumb by an extendedpeptide (Fig. 2B).

The crystal structure of S. cerevisiae Pol� lacked 120 C-terminal residues,which, although unnecessary for polymerase activity in vitro, are likely to beessential for protein–protein interactions with accessory factors, such asPCNA (see below). The corresponding C-terminal domain in human Pol�is much longer (280 vs. 120 amino acids [aa]).

The crystal structure of Pol� was determined in the absence of DNA, butthe location of DNA within the polymerase was determined by fitting thecrystal structure of Pol� to the previously reported template-primer-ddNTP ternary complex of T7 DNA polymerase (Trincao et al., 2001).Using such an approach, it was shown that the binding pocket of thepolymerase around the templating base is likely to be open or extended,thereby providing an explanation for the low fidelity of the polymeraseand suggesting a mechanism by which altered bases in the template can be

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accommodated within the active site. In particular, modeling of a cova-lently linked thymine dimer into the active site of Pol� suggests it hasthe capacity to accommodate two templating residues. Support for thismodel comes from recent structural studies of Dpo4, a Y-family polymerasefrom Sulfolobus solfataricus, whose structure has been solved in a ternarycomplex with CDP-containing DNA and an incoming nucleotide (Linget al., 2003). The Dpo4-CPD structure clearly showed that the covalentlylinked CPD was readily accommodated within the active site of Dpo4.Modeling of Pol� onto the Dpo4 structure further suggested thatthe finger, thumb, and LF polymerase domains undergo a conformationalchange from an ‘‘open,’’ DNA-free complex to a ‘‘closed,’’ CPD-containing complex, which facilitates efficient bypass of the CPD (Linget al., 2003).

The possibility that Pol� retains both thymine bases of the CPD withinits active site and directs incorporation of the correct nucleotides by usingthe intrinsic base-pairing ability of the lesion is also supported by kineticstudies (Washington et al., 2003b). Furthermore, structural and biochemi-cal data suggest that intact Watson–Crick base pairing rather than thegeometric fit of the incoming nucleotide with the templating base withinthe polymerase active site plays a crucial role in the efficiency and fidelityof DNA synthesis by Pol�. Thus, Pol� accommodates a CPD lesion becauseof the openness and flexibility of the active site, but also because Watson–Crick base pairing, although weakened, remains intact at the CPD. Simi-larly, yeast Pol� readily traverses through 8-oxoG with the preferentialformation of a C:8-oxoG base pair, which retains the three hydrogen bondsfound in a normal C:G base pair (Haracska et al., 2000). In contrast to theCPD and 8-oxoG, an abasic site or 6-4PP lesion represents a strong obstacleto replication by Pol�, as any hydrogen bonding potential is completelyabsent at an abasic site and is significantly disrupted at the 30 side of the6-4PP. The importance of the ability to form normal hydrogen bonds duringPol�-catalyzed DNA synthesis is further supported by the drastic decrease ininsertion fidelity of yeast Pol� in replication reactions with difluorotoluene,a nonpolar isosteric analog of thymine that is unable to form Watson–Crickhydrogen bonds with adenine but is virtually identical to dT in shape, size,and conformation (Washington et al., 2003a).

VI. REGULATION AND LOCALIZATION OF POL� AND POL�

The human POLH gene is expressed in all tissues, with small increases inexpression in proliferating tissues such as testis, thymus, and skin, and alsoin the liver (Yamada et al., 2000). In mouse cells, Pol� expression increasedwhen serum-starved cells were stimulated to proliferate (Yamada et al.,

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2000). However, Pol� is not required for normal DNA replication, as XP-Vcells proliferate normally in the absence of DNA damage (Lehmann et al.,1975). Although the level of POLH mRNA decreases temporarily afterUV irradiation (Yamada et al., 2000), protein levels are unaffected(P. Kannouche and ARL, unpublished observations). Both human andmurine POLI are similarly expressed in all tissues, with the highest level ofexpression observed in postmeiotic spermatids from testis (Frank et al.,2001; McDonald et al., 1999).

To gain insight into how Pol� and Pol� function inside the cell, localiza-tion studies have been carried out using eGFP-tagged polymerase con-structs (Kannouche et al., 2001, 2002). This work showed that bothpolymerases were localized exclusively in the nucleus and were uniformlydistributed through the nucleus in the majority of cells in an asynchro-nous culture of SV40-transformed human fibroblasts. However, in a smallproportion of the cells (typically 15%), the polymerases accumulated inmany bright foci, where they colocalized both with each other and withPCNA. These foci are thought to be replication factories, in which DNAreplication is occurring. After treatment with UV irradiation, the numberof cells containing bright foci of colocalizing Pol�, Pol�, and PCNAincreased up to about 70% of the population (Fig. 3). It is believed thatthe number of foci increases because UV-induced DNA damage blocksprogression of the replication forks and slows down cell passage through Sphase. The resulting gradual accumulation of cells in S phase accounts forthe increase in the number of cells with foci containing Pol�, Pol�, andPCNA, rather than DNA damage-induced relocalization of these proteins,as it was initially postulated. Consistent with this idea, accumulation ofnuclei with foci containing Pol� and Pol� has also been observed after

FIG. 3. Colocalization of Pols � and � in response to UV irradiation. Foci formationafter UV damage is shown for Pol� (red) (left) and Pol� (green) (middle). Bothpolymerases form foci in the same time and space within a cell following UV irradiation,indicating that the accumulation of Pol� and Pol� at replication forks stalled at sites ofUV damage is coordinated in vivo. The colocalization of Pol� and Pol� is indicated by ayellow pattern in the merged figure (right). This figure is reproduced with permissionfrom Kannouche et al., 2002.

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other treatments that block progression of the replication fork, such asmethyl methanesulfonate or N-acetoxy-acetylaminofluorene, which gener-ate DNA damage, and hydroxyurea, which arrests cells in S phase, withoutcausing DNA damage. The localization of Pol� in replication foci is partlydependent on the presence of Pol� because in XP-V cells, Pol� remainsnuclear, but its accumulation in replication foci is reduced approximatelythree- to six-fold (Kannouche et al., 2002).

Deletion analysis has identified the domains required for correct locali-zation of the polymerases (Fig. 2A). For Pol�, the last 70 aa, containing abipartite nuclear localization signal, are sufficient for nuclear localization,but not for accumulation into foci. However, the last 120 aa are sufficientfor localization both in the nucleus and in nuclear foci (Kannouche et al.,2001). This 120-aa region contains a C2H2 zinc finger motif and, at theextreme C terminus, a PCNA-binding motif (Fig. 2A). Both of these arerequired for localization into foci (P. Kannouche and ARL, unpublishedresults). Pol� does not contain any obvious motifs in the unconserved C-terminal part of the protein, and similar analyses were much less clear-cut.Localization into the nucleus required sequences contained within aa219–451 at the end of the polymerase domain and the beginning of thenonconserved domain, whereas foci formation required the C-terminal220 aa (Fig. 2A; Kannouche et al., 2002). Pol� and Pol� were shown tointeract directly, and this same C-terminal 220 aa of Pol� were required forthe interaction with Pol�. Taken together with the partial dependence onPol� for foci formation, these data suggest that Pol� helps transport Pol�into nuclear foci.

The cellular role of aa 430–600 of Pol�, which is poorly conservedamong the Pol� orthologs, is currently unknown.

VII. MUTATIONS IN POL� IN XP VARIANTS

The two initial studies in which POLH was discovered to be the genedefective in XP-V cells also identified several nonsense mutations inhuman patients with XP-V ( Johnson et al., 1999a; Masutani et al., 1999).Subsequent work identified many further mutations in XP-V patients(Broughton et al., 2002; Itoh et al., 2000), and these are summarized inFigure 4. The majority of the mutations cause truncations in Pol� thatare located within the conserved polymerase domain found in all Y-familypolymerases and that are likely to produce a completely inactive protein.This suggests that Pol� is not required for human life. A few truncat-ing mutations are located close to the C terminus, outside of the polymer-ase domain. Extracts from these cells have TLS activity characteristic forPol� (Broughton et al., 2002), but we anticipate that the truncated Pol�

FIG. 4. Locations of mutations in POLH that lead to the Xeroderma pigmentosum variant (XP-V) phenotype. The locations of the aminoacid changes identified in patients with XP-V are shown relative to the different domains of the polymerase shown in Fig. 2B. The structuraldomains of Pol� are shown in blue (finger, F), red (palm, P), green (thumb, T), and purple (little finger, LF) in the central part of the diagram.The nuclear localization signal (NLS) and the domain involved in localizing Pol� into replication foci (URS) are also indicated in black andbrown, respectively. Cell strains are designated in boxes. Subscripts 1 and 2 specify different alleles. Deletions are shown using horizontal lines.Single amino acid changes are shown in the upper part of the diagram, and truncations are shown in the lower part.

218V

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DNA POLYMERASES � AND � 219

molecules would probably not reside in the replisomes, as they are missingthe C-terminal domain required for localization in the nucleus and innuclear foci.

Several missense mutations, mostly located within the conserved catalyt-ic domain, have also been identified (Fig. 4, top). These were modeledonto the three-dimensional structure of Dpo4 from Sulfolobus solfataricus.Two of the mutated amino acids, G263 and R361, are involved in interac-tions with the DNA, whereas the other mutations are likely to disrupt theconformations of the different domains (Broughton et al., 2002). Interest-ingly, a Japanese patient is a compound heterozygote for two mutations,K535E and K589T, both located in the poorly conserved central domainbetween the polymerase and localization domains (Itoh et al., 2000). K535is, however, in a run of nine aa conserved in mammalian species of Pol�,and K589 is conserved in mouse and rat, demonstrating importantfunctions for this part of the protein.

As mentioned above, the severely truncating mutations suggest that Pol�is not an essential gene in humans, and in support of this idea, a trans-genic mouse lacking Pol� has recently been generated (Fumio Hanaoka,personal communication).

VIII. POLS � AND � AND THE POLYMERASE SWITCH: INTERACTIONS WITH

PCNA AND REV1

A topic of major interest that is also discussed in more detail in otherchapters of this book is the mechanism by which the replicative DNApolymerase is displaced at a replication fork blocked by DNA damage andis replaced with a TLS polymerase. All three of the E. coli TLS polymeraseshave motifs involved in the interaction with the sliding clamp � that helpsto recruit polymerases to their site of action (Becherel et al., 2002). Ineukaryotic cells, proliferating cell nuclear antigen (PCNA) plays the roleof a sliding clamp, and analyses of the primary aa sequences of Pols � and �reveal putative PCNA-binding motifs. Yeast two-hybrid assays confirmedthat there is a physical interaction between Pol� and PCNA in vivo(Haracska et al., 2001a); and in vitro replication assays revealed that thecatalytic activities of both Pol� and Pol� on undamaged and damaged DNAtemplates were increased by the presence of PCNA along with clamploader, replication factor C (RFC), and RPA (replication factor A)(Haracska et al., 2001a,b).

As discussed in detail in Chapter 10, PCNA becomes both mono- andpolyubiquitinated in yeast cells following treatment with MMS (Hoegeet al., 2002). Based on these findings, it was postulated that PCNA modifi-cation was involved in the polymerase switch (Hoege et al., 2002), and this

220 VAISMAN ET AL.

idea was supported by genetic evidence showing that ubiquitination ofPCNA was epistatic with POL30 (Stelter and Ulrich, 2003). In mammaliancells, in response to UV irradiation, PCNA is only monoubiquitinated, andthis ubiquitination is not dependent on NER or on Pol�. The monoubi-quitinated PCNA is found exclusively in chromatin, where it interactsphysically and specifically with Pol� (Kannouche et al., 2004). This inter-action, detected by coimmunoprecipitation, was found only with themodified form of PCNA. No interaction was detected with unmodifiedPCNA in cell extracts. The increased affinity for Pol� of monoubiquiti-nated PCNA generated at blocked replication forks therefore provides anattractive mechanism for mediating the polymerase switch at the sites ofDNA damage.

As discussed at the beginning of this chapter, Rev1 is phylogeneticallyrelated to the Y-family polymerases, but it primarily inserts dCMP, andnot the three remaining dNTPs. Recently, several groups have inde-pendently demonstrated that Pol� aa residues 370–492 interact with theC-terminal 100 aa of Rev1 (Fig. 2A) (Guo et al., 2003; Ohashi et al., 2004;A. Tissier et al., 2004). The same C-terminal fragment of Rev1 also inter-acts with Pol�, Pol� (see Chapter 9), and Rev7 (see Chapter 6) (Guo et al.,2003; Ohashi et al., 2004; Tissier et al., 2004). Revl, like Pols � and �, islocalized in replication factories (Tissier et al., 2004). These findingssuggest that Revl might play a role as a molecular scaffold during thebypass of DNA lesions, facilitating aspects of the polymerase switchingmechanisms.

IX. PROTECTION FROM CELLULAR EFFECTS OF DNA DAMAGE

Early work showed that, in contrast to NER-defective XP cells, XP-Vfibroblasts were only marginally sensitive to killing by UV light (althoughthey are specifically sensitized by caffeine). They were, however, like NER-deficient XP cells, highly sensitive to the induction of mutations by UVlight (Maher et al., 1976). We may now interpret these data as suggestingthat TLS by Pol� plays only a minor role in protecting human cells fromkilling by UV light but has a major role in safeguarding us from mutations.This is absolutely consistent with the finding that Pol� in most instancesinserts the ‘‘correct’’ nucleotides opposite CPDs (discussed above, insection III), and that in its absence, the substituting mechanism is muchmore error prone. In recent work supporting these findings, cell lineshave been derived in which XP-V cells stably express a transfected POLHgene. UV mutagenesis was measured using two different systems, and themutant frequency was restored to normal levels in the Pol�-transfectedcells (Stary et al., 2003; Taylor et al., 2003).

DNA POLYMERASES � AND � 221

The UV mutation spectra in XP-V cells differ significantly from those innormal or NER-defective XP cells. In particular, whereas most UV-inducedmutations in the latter two cell types are C to T transitions, in XP-V cells thereis a greater proportion of mutations at thymines (Wang et al., 1993). Thus, inthe absence of Pol�, the substituting enzymes make more errors at A or Tresidues. On the basis of biochemical data, it seems reasonable to hypothe-size that Pol� might be the enzyme responsible for such phenotype. Althoughthe ability of Pol� to replicate CPD-containing templates is much lower thanthat of Pol�, it is higher than that of any other known eukaryotic polymeraseand is very similar to that observed for the well-characterized E. coli transle-sion polymerase, PolV (Tang et al., 2000; Vaisman et al., 2002). In addition,the highly error-prone behavior of Pol� at template T (Tissier et al., 2000a),and the increased accuracy of nucleotide incorporation opposite deami-nated cytosines compared to other polymerases (Vaisman and Woodgate,2001), is consistent with the mutation spectra observed in XP-V cells.

X. ROLES OF POL� AND POL� IN SOMATIC HYPERMUTATION

Somatic hypermutation (SHM) is one of the mechanisms by whichantibody diversity is generated and is the subject of Chapter 11. Modelsfor the mechanism of SHM implicate an error-prone polymerase, andseveral groups have investigated the involvement of different Y-familypolymerases in this process. In lymphocytes from three XP-V patients,the frequency of SHM was normal but the types of base changes weredifferent: There was a decrease in mutations at A and T and a concomitantrise in mutations at G and C. These results suggest that more than onepolymerase contributes to SHM, and that if one is absent, others compen-sate. The data suggest that Pol� is involved in generating errors that occurpredominantly at A and T and that another polymerase may preferentiallygenerate errors opposite G and C (Zeng et al., 2001). In support of thisidea, the SHM hotspots at A and T residues correlate with the Pol� errorspectrum (Rogozin et al., 2001). With regard to Pol�, the data presentlyavailable are inconclusive. Targeted inactivation of the POLI gene in theBurkitt’s lymphoma BL2 cell line abolished SHM, suggesting that Pol� isabsolutely required for SHM in these cells (Faili et al., 2002). In contrast,the frequency and spectrum of SHM were indistinguishable in a129-derived mouse strain that contains a stop codon early in the Poli geneand in Pol�þ mice (McDonald et al., 2003). These latter data suggest thatPol� is dispensable for SHM. However, both systems have their peculia-rities, and so extrapolation to the human population must be treated withcaution. It therefore remains plausible that Pol�, like Pol�, may play a rolein SHM, but that the various polymerases have redundant functions.

222 VAISMAN ET AL.

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