Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase η from Arabidopsis thaliana

Mutation Research 601 (2006) 51–60

Analysis of UV-induced mutation spectra in Escherichia coli by DNA polymerase ␩ from Arabidopsis thaliana Mar´ıa Jes´us Santiago 1 , Encarna Alejandre-Dur´an 1 , Manuel Ruiz-Rubio ∗ Departamento de Gen´etica, Facultad de Ciencias, Edificio Gregor Mendel, Campus Rabanales, Universidad de C´ordoba, Spain Received 16 December 2005; received in revised form 15 May 2006; accepted 26 May 2006 Available online 20 July 2006

Abstract DNA polymerase ␩ belongs to the Y-family of DNA polymerases, enzymes that are able to synthesize past template lesions that block replication fork progression. This polymerase accurately bypasses UV-associated cis–syn cyclobutane thymine dimers in vitro and therefore may contributes to resistance against sunlight in vivo, both ameliorating survival and decreasing the level of mutagenesis. We cloned and sequenced a cDNA from Arabidopsis thaliana which encodes a protein containing several sequence motifs characteristics of Pol␩ homologues, including a highly conserved sequence reported to be present in the active site of the Y-family DNA polymerases. The gene, named AtPOLH, contains 14 exons and 13 introns and is expressed in different plant tissues. A strain from Saccharomyces cerevisiae, deficient in Pol␩ activity, was transformed with a yeast expression plasmid containing the AtPOLH cDNA. The rate of survival to UV irradiation in the transformed mutant increased to similar values of the wild type yeast strain, showing that AtPOLH encodes a functional protein. In addition, when AtPOLH is expressed in Escherichia coli, a change in the mutational spectra is detected when bacteria are irradiated with UV light. This observation might indicate that AtPOLH could compete with DNA polymerase V and then bypass cyclobutane pyrimidine dimers incorporating two adenylates. © 2006 Published by Elsevier B.V. Keywords: DNA polymerase ␩; UV mutagenesis; tRNA suppressor mutations; Arabidopsis thaliana; Escherichia coli

1. Introduction DNA is damaged by many endogenous and exogenous agents. The altered nucleotides in DNA are removed by DNA repair mechanisms, recovering the original DNA chemical structure. However, some of the injured nucleotides may escape repair and present a block to continue DNA replication by DNA polymerases. Consequently, the replication fork arrests and further mechanisms are necessary to cope with full DNA

∗ 1

Corresponding author. Tel.: +34 957218979; fax: +34 957212082. E-mail address: [email protected] (M. Ruiz-Rubio). These authors contribute equally to this work.

0027-5107/$ – see front matter © 2006 Published by Elsevier B.V. doi:10.1016/j.mrfmmm.2006.05.036

synthesis in the presence of the damaged nucleotides [1]. One of these mechanisms depends on the Y-family of DNA polymerases, enzymes which have the capability to carry out DNA translesion synthesis (TLS) across damaged or missing bases. In bacteria their expression has been shown to be induced by DNA damage [2]. The TLS DNA polymerases do not have a 3 –5 exonuclease activity, as a result they achieve replication with low fidelity through an undamaged template. Several excellent reviews has been written about this topic [3–7]. The primary sequences of Y-family polymerases have five conserved motifs confined to the amino (N)-terminal part of the protein. The family includes five subfamilies [7] represented by (i) Escherichia coli DNA polymerase IV-dinB [8], (ii) E. coli DNA polymerase V-umuC [9,10],

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(iii) eukaryotic DNA polymerase ␩ [11,12], (iv) Rev1, an eukaryote DNA-dependent dCMP transferase [13] and (v) Pol ␫, which has the lowest fidelity of any eukaryotic DNA polymerase studied to date [3,14,15]. UmuC-like polymerases, that are required for the major mechanism of damage-inducible mutagenesis, have been found only in bacteria, but proteins of the DinB subfamily have been found in bacteria, archaean, plants and other eukaryotes [16,17]. In yeast and human cells, bypass of cyclobutane pyrimidine dimers (CPDs) is thought to be carried out by Pol␩. Unlike PolV, Pol␩ is able to promote error-free replication through the UV-induced cis–syn dipyrimidine photoproducts [18–20]. Purified human Pol␩ synthesizes past CPDs thymine–thymine (T–T) dimers in vitro, with efficiency comparable with that observed for undamaged DNA, inserting primarily adenines opposite the lesions [21,22]. Additionally, human Pol␩ is able to replicate DNA across a wide spectrum of lesions with efficiency and accuracy, depending on both the damage and the sequence around the altered nucleotides [19,22–24]. Similar to other TLS polymerases, it shows low fidelity when replicating undamaged DNA [25], which suggests that it might have other functions in addition to bypass of DNA damage. It has been proposed that human Pol␩ and other TLS polymerases might be implicated in somatic hypermutation of the immunoglobulin variable region genes [3,26,27]. Plants can reverse UV-induced DNA damage by photoreactivation or remove it via nucleotide excision repair [28–31]. In addition, plants may tolerate UV photoproducts via TLS [30]; recently a DinB-like polymerase from Arabidopsis thaliana has been described [16]. To obtain a more comprehensive understanding of TLS mechanisms in plants we have carry out the identification, basic characterization, and expression analysis of AtPolH, a gene coding one Y-family DNA polymerase, that is homolog to yeast, human and other DNA polymerase ␩. We also describe the change of UV-induced mutation spectra in bacteria when they produce AtPOLH. Thus, the analysis of prototrophic mutants of bacteria carrying the argE3 allele and an amber suppressor mutation, using nonsense bacteriophage T4 strains [32–34], allowed to distinguish between true revertants (backmutations) and ochre suppressor mutations. We found that the expression of AtPOLH in E. coli decrease backmutations and increase the number of specific types of ochre tRNA suppressors. These results may suggest that in the bacterial system, AtPOLH could compete with DNA polymerase V and bypass cyclobutane pyrimidine dimers incorporating two adenylates.

2. Materials and methods 2.1. Yeast, bacterial and phage strains. Plasmids The Saccharomyces cerevisiae strains EH150 (MATa lys2dBgl trp1-his3-200 ura3-52 ade2-1 gal mal CUPr) and YLB30 (like EH150 but carrying a rad30::HIS3 allele), derived from strain YNN281 (Yeast Genetic Stock Centre). They were kindly provided by Dr. E. Heindereich (Medical University of Vienna, Institute of Cancer Research, Austria). E. coli strains used for cloning and transformation were XL1-Blue MRF ((mrcA)183, (mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac[F proAB lacIq lacZ ΔM15, Tn10 (Tetr )]) and XLOLR ((mcrA)183,  (mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1, gyrA96, relA1, lac[F proAB lacIq lacZ M15, Tn10 (Tetr )]) from Stratagene, and DH5␣ (supE44, ΔlacU169, (φ80 lacZΔM15), hsdR17, recA1, endA1, gyrA96, thi-1, relA1) from Life Technologies, Inc. The bacterial strain used for mutagenesis, IC1141 (uvrB5, thr-1, proA2, hisG4 (Oc), argE3 (Oc), thi-1, lacY1, galK2, srl::Tn10, malE7::Tn5, sfiB, str-31, sup-37) [35], and the strains of T4 phage employed for mutational analysis, NG75 (amber mutant), PS205 (ochre mutant) and NG273 (ochre mutant), have been described previously [32,34]. The bacterial plasmids used were pSE420 (Invitrogen), pMALc2X (New England BioLabs), pET28c (Novagen) and the shuttle yeast plasmid pYPGE15 [36]. 2.2. Cloning and subcloning of ATPOLH cDNA An A. thaliana cDNA library CD4-15 (Arabidopsis Biological Resource Center, OH, USA) was screened by in situ plaque hybridisation. Two positive halos were isolated using a digoxigenin-dUTP (Roche Applied Science)-labelled probe obtained by PCR of genomic DNA from A. thaliana. The probe had a length of 1137 bp and the primers used were AtPoletaF2 and AtpoletaR2 (Table 1). The corresponding clones were excised in vivo into the phagemid form. Plasmid DNA was purified with a mini-plasmid purification kit (Qiagen) and the cDNAs were sequenced for both strands using AtPoletaF1, R1, F2, R2 and F6 primers (Table 1). One of the plasmids contained the putative coding sequence of Pol␩ from A. thaliana and it was named pBS-AtPOLH. The cDNA corresponding to putative AtPOLH of pBSAtPOLH was subcloned in pSE420 expression plasmid. PCR with Pfx DNA polymerase was used to amplify the coding region. The primers AtPoleta-NcoI and AtPoleta-NotI (Table 1) were used to introduce an NcoI site at the 5 end of the ORF and a NotI site within the 3 UTR and then ligated into pSE420 vector. A SalI and PstI digestion fragment of pSE420-ATPOLH containing the cDNA of putative AtPOLH, except the 48 bp corresponding to the first 16 amino acids, was subcloned in frame at the C-terminus of the E. coli maltose-binding protein (MBP) of the pMAL-c2X expression vector, to construct the pMAL-ATPOLH recombinant plasmid.

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Table 1 Primers used for PCR and RT-PCR Name

Sequence

AtPoletaF1 AtPoletaR1 AtPoletaF2 AtPoletaR2 AtPoletaF6 AtPoleta-NcoI AtPoleta-NotI AtPoleta-NcoIb AtPoleta-XhoI AtActinF1 AtActinR2 MutetaF MutetaR

5 -TCTCTCTCCCTCTGATTTCTGCGGAG-3 5 -CGGTGTTAAACTGCTTATTGGTGCGT-3 5 -TCGACATGGATTGCTTCTACGTTCAAG-3 5 -GGGCTATGCCAGCAGAACAAGTAAAC-3 5 -AGAAACTCAAGCAGCAATGCCTGAAG-3 5 -ATATCCATGGCGGTAGCGAGACCGGAAGC-3 5 -TACAGCGGCCGCCAGAGACAAAGGAGCTTA-3 5 -ATAGCCATGGCGGTAGCGAGACCGGAAGCA-3 5 -CGGCCTCGAGCTGCTTATTGGTGCGTAGGA-3 5 -AAAGGATGCTTATGTTGGCG-3 5 -GAAAGAGTAACCACGCTCGG-3 5 -GGGCTTCGATTGCGGCCGCGTATCTTGACC-3 5 -GGTCAAGATACGCGGCCGCAATCGAAGCCC-3

On the other hand, AtPOLH cDNA from pSE420-ATPOLH was subcloned in pET28c amplifying by PCR with Pfx DNA polymerase using specific primers AtPoleta-NcoIb and AtPoleta-XhoI (Table 1). The amplified fragment from pSE420-ATPOLH containing the coding region was digested with both NcoI and XhoI enzymes and finally inserted into pET28c vector to obtain the recombinant plasmid pETATPOLH. The sequence of the insert was confirmed by DNA sequencing analysis. Finally, an XbaI and XhoI digestion fragment from pETATPOLH plasmid containing the cDNA of AtPOLH, was inserted in plasmid pYPGE15 resulting the recombinant plasmid pYPGAtPOLH to study complementation in yeast. 2.3. Construction of a mutated AtPOLH An inactive AtPOLH protein was engineered by PCR using the primers MutetaF and MutetaR (Table 1). In this AtPOLHmut the conserved SIDEV site of motif III was changed by SIAAA. AtPOLHmut was inserted in pYPGE15 plasmid resulting the recombinant plasmid pYPGAtPOLHmut. 2.4. Sequence analysis Identification of potential AtPOLH homologues was carried out by a BLAST search at the National Center for Biotechnology Information (NCBI). Similarity of the sequences retrieved from the BLAST search was analyzed by multiple sequence alignment with the Bioedit program [37]. Sequences were aligned using PILEUP and a neighbour-joining distance tree was constructed using CLUSTAL W [38]. 2.5. Plant material and growth conditions Seeds of A. thaliana plants (ecotype Columbia) were placed on plastic pots with universal substrate (Composana® , Compo GmbH, Germany) and covered with clear plastic covers. The pots were placed in a cold room at 4 ◦ C for 2 days in the dark to synchronize germination, and then moved to a grown room at

23 ◦ C with a 16-h light/8-h dark cycle photoperiod. After 3–4 days, the plastic covers were removed. 2.6. Reverse transcription-PCR (RT-PCR) analysis of AtPOLH expression Total RNA from different plant tissues, of around 4 weeks old, were isolated as described [39] and treated with RNAsefree DNAse (Pharmacia-Biotech). RT-PCR was performed using the “Retrotools” cDNA/DNA kit (Biotools) following the manufacture’s instructions. Oligo dT and primers AtPoletaF6 and AtPoletaR1 (Table 1) were used. The amplification was as follows: denaturation at 94 ◦ C for 30 s, annealing at 55 ◦ C for 30 s, and extension at 72 ◦ C for 90 s. An initial denaturation step of 5 min at 94 ◦ C and a final elongation step at 72 ◦ C for 10 min were performed. The AtActin-1 PCR product was utilized as a constitutive control using the primers AtActinF1 y AtActinR2 (Table 1). 2.7. Yeast growth and UV irradiation Yeast cells were transformed with pYPGE15 or pYPGATPOLH. Stationary phase yeast liquid cultures, in complete minimal medium (without uracil to select transformed yeast), were harvested and resuspended in water at 106 cells/ml; 10 ml of this suspension were placed into a 60 mm diameter plastic Petri dish and used for irradiation. The UV doses used at 254 nm (Vilvert Lourtmat lamp); were 50 and 80 J/m2 at a dose rate of 4 J/m2 /s. The incident fluence was measured with a radiometer VLX254. Appropriate dilutions were plated on complete minimal medium (with 1.5% of agar) for viability determination. Plates were incubated at 30 ◦ C and colonies were counted after 3 days for viability. At least three independent experiments were achieved. 2.8. Bacterial growth and UV mutagenesis Bacteria IC1141 transformed with pMALc2X or pMALATPOLH were grown at 37 ◦ C on minimal medium (glucose

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2%, thiamine 2 ␮g/ml, histidine, arginine, threonine and proline 50 ␮g/ml) with carbenicillin (50 ␮g/ml) and tetracycline (10 ␮g/ml) until the absorbance was 0.4 at 600 nm. The culture was induced by adding IPTG to 0.3 mM for 2 h. Then, the cells were collected by centrifugation and resuspended in 10 mM MgSO4 at 108 /ml; 10 ml of this suspension were placed into a 60 mm diameter plastic Petri dish and used for irradiation. The mutagenesis was performed using different doses of UV at 254 nm (0, 2 and 4 J/m2 ). Aliquots were removed and appropriate dilutions were plated on minimal medium for determination of viable bacteria. For selection of Arg+ revertants, the same minimal medium without arginine was used, but a small amount of arginine (2 ␮g/ml) was added. The colonies were counted after 2 days at 37 ◦ C. The Arg+ mutant types were determined by tests with nonsense defective bacteriophage T4 (see below). At least three independent experiments were performed. The results were analyzed by χ2 -test [40]. 2.9. Distinguishing mutant types by bacteriophage tests Prototrophic cells were tested with nonsense defective bacteriophage T4 to determine whether they result from backmutations or specific suppressor mutations [34]. In this study three different T4 strains were used to determine patterns of growth and lysis (+) or no growth (−), with individual prototrophic isolated on Luria broth agar [41]. Bacteriophage NG75, PS205 and NG273, respectively gave patterns (+−−) for backmutation, (−+−) for converted glutamine tRNA suppressor mutation, (++−) for de novo glutamine tRNA ochre suppressor mutation and (+−+) for de novo tyrosine tRNA ochre suppressor mutation. The pattern for backmutation was the same as that for parental cells, which is known to contain a glutamine tRNA amber suppressor mutation (supE44) [42].

3. Results 3.1. Isolation of a clone containing a cDNA encoding DNA polymerase η from A. thaliana The A. thaliana genome sequence [43] was screened, and a potential orthologue of S. cerevisiae and Homo sapiens Pol␩ coding gene, was identified on chromosome 5. Using specific primers (see Section 2), a 1.1 kb genomic fragment of the putative gene from Arabidopsis obtained by PCR, was used as a probe to screen an A. thaliana cDNA library. Two positives clones were obtained, and one of them contained a putative AtPOLH full-length coding cDNA of 2139 pb long. Comparison of the genomic and cDNA sequences reveals 14 exons and 13 introns (EMBL Accession No. AJ416380.1) and confirmed that the coding region contained a 2019 bp open reading frame encoding a predicted 672 amino acid protein with a molecular mass of 73.9 kDa. A database search with the deduced amino acid sequence of the puta-

tive AtPOLH protein reveals a high degree of similarity to Pol␩ from various organisms, including yeast, insects, nematodes and mammals. Alignment of the amino acid sequences shows the presence of five conserved motifs, I–V in the N-terminus (Fig. 1), a distinctive feature of the Y-family [44]. Of particular interest is the motif III, which contains the highly conserved sequence ASIDE and is involved in the DNA polymerase active site [45]. The carboxy(C)-terminal sequence of AtPOLH, although less conserved respect the other DNA polymerase ␩ (alignment not showed), posses the sequence QRELRSFL at position 636–643. It is very similar to QxxLxxFF that is present in most DNA polymerases. This amino acid sequence is required for binding to the proliferating cell nuclear antigen (PCNA), which mediates processive DNA synthesis and damage bypass [46]. The interaction between yeast Pol␩ and PCNA has been shown to be required for in vivo function of this polymerase [47], and has been also identified in human [48]. In yeast and human, mutations of this consensus motif result in a phenotype equivalent to that of a null mutation [47]. The C-terminus of Pol␩ may also function in other specific protein–protein interactions [49]. 3.2. Expression of ATPOLH in A. thaliana To confirm that the AtPOLH mRNA is indeed present in vivo, we performed RT-PCR with total RNA obtained from different A. thaliana tissues and gene specific primers. Fig. 2 shows the presence of an amplified fragment with a size close to that expected (500 pb) from the AtPOLH cDNA sequence, in all the tissues tested. The control reaction performed with genomic DNA as template, yielded a band of about 800 pb (expected 831 pb) that includes the two last introns absent in the cDNA. 3.3. Functional complementation by ATPOLH of a S. cerevisiae strain deficient in DNA polymerase η The AtPOLH gene from A. thaliana was checked for its ability to complement the Pol␩ minus phenotype of a S. cerevisiae strain. Survival curves of strain EH150 of S. cerevisiae obtained with 254 nm wavelength UV light irradiation, indicated that around 65% and 55% of the yeast cells survived with a fluence of 50 J/m2 and 80 J/m2 , respectively (Fig. 3). The same strain harbouring a rad30 defective mutation, YLB30 (rad30::HIS3), was twice more sensitive to UV light radiation (Fig. 3). The yeast rad30 deficient strain transformed with pYPGAtPOLH restored the survival, while

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Fig. 1. (A) Amino acid sequence alignment of N-terminus corresponding to several Pol␩ from different species. All sequences were retrieved from GenBank. Accession numbers are: Arabidopsis thaliana AJ416380 (672 aa); Caenorhabditis elegans NP 497480 (603 aa); Drosophila melanogaster BAB20905 (885 aa); Homo sapiens AAD43810 (713 aa); Mus musculus NP 109640 (694 aa); Saccharomyces cerevisiae NP 010707 (632 aa); Schizosaccharomyces pombe CAA16862 (872 aa). (B) Phylogenetic analysis of Pol␩ homologues.

Fig. 2. Expression of AtPOLH in different plant tissues. RT-PCR was performed using specific primers from AtPOLH and AtActin-1 on total RNA from multiple plant tissues. F: flowers, RL: rosette leaves, CL: cauline leaves, R: roots, Si: siliques, St: stems and G: control genomic DNA.

the yeast mutant transformed with the pYPGE15 plasmid, as control, did not complement this deficiency (Fig. 3). An inactive AtPOLH protein (AtPOLHmut), in which the conserved ASIDEV site of motif III was changed by ASIAAA, was used as a negative control. Other authors have found that ASIDEV site is essential for polymerase activity [50]. As expected, rad30 mutation was not complemented by pYPGAtPOLHmut (Fig. 3). This result established that AtPOLH gene from A. thaliana complements the phenotype of UV sensitivity of the rad30 defective mutation in S. cerevisiae and showed that AtPOLH encodes a functional protein.

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Fig. 3. Functional complementation of S. cerevisiae Pol␩ deficient mutant with AtPOLH. Survival to different doses of ultraviolet light (UV) of wild type strain EH150 S. cerevisiae (), YLB30 S. cerevisiae Pol␩ deficient (), YLB30 transformed with pYPGE15 (䊉), YLB30 transformed with pYPGAtPOLH () and YLB30 transformed with pYPGAtPOLHmut (). Each value is the mean of six independent experiments.

Additional experiments (data not shown), proved that canavanine resistance mutation frequency induced by UV in rad30 S. crevisiae strain, was restored to the wild type level by AtPOLH but not by AtPOLHmut. 3.4. Effect of AtPOLH on UV-induced mutagenesis in E. coli To test whether AtPOLH is functional in E. coli, an expression plasmid overproducing this TLS polymerase, pMAL-AtPOLH, was introduced in the strain IC1141. This E. coli strain is deficient in nucleotide excision repair and has an argE3 ochre mutation. Bacteria were UV irradiated and the survival and the UV-induced mutation frequency were determined. These parameters were similar in bacteria transformed with pMAL-AtPOLH and with pMALc2X (data no showed). To see if there was a change in the mutation spectra, the types of arginine prototrophic mutants were analyzed using nonsense defective bacteriophage T4. 3.4.1. Decrease of UV-induced backmutations in bacteria expressing AtPOLH Bacteria UV irradiated carrying pMAL-AtPOLH showed a decrease in the number of backmutations respect of the control bacteria with pMALc2X (Fig. 4).

Fig. 4. Yields of specific mutants types by UV mutagenesis in Escherichia coli IC1141 strain transformed with pMAL (pMAL) or transformed with pMAL-AtPOLH (pMALeta). Mutant types: backmutation (pMAL R and pMALeta R), de novo glutamine tRNA ochre suppressor (pMAL dG and pMALeta dG), and converted glutamine tRNA suppressor (pMAL cG and pMALeta cG). The mutants were determined by tests with nonsense defective bacteriophage T4 (see Section 2). *** p < 0.005, ns (p > 0.05).

This observation suggests, according with the target sequence to produce backmutations (Table 2), that while PolV introduces a G in front of a T contained in a TT pyrimidine dimer, AtPOLH introduces an A, reducing this type of specific mutants. This result agrees with the report from other authors [22,51], which describe that in vitro, Pol␩ efficiently incorporates two adenylates opposite the cis–syn TT dimer and therefore it performs an error-free TLS.

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Table 2 Pattern of T4 phage strains growth on different types of mutants Target sequence

-TAA-, -ATT-TTTTGAT-, -AAAACTA-TTCTAAT-, -AAGATTA-CTGTAAA-, -GACATTTa

Possible mutation (in bacterial DNA)

Sense codon -TTA-, -AAT-TTA-, -AAT-TTA-, -AAT-

Mutant types

Back mutant De novo Gln Converted (Gln, amber to ochre) De novo Tyr

T4 phage straina NG75

P205

NG273

+ + − +

− + + −

− − − +

Phage growth (+) or not growth (−) on arg+ mutant types of E. coli IC1141 strain.

3.4.2. Increase of UV-induced de novo and converted tRNA Gln ochre suppressor mutations in bacteria expressing AtPOLH The number of arginine prototrophic mutants which are de novo tRNA Gln ochre suppressors and converted tRNA Gln from amber to ochre suppressors, increased by UV light when AtPOLH is present with respect to the control (Fig. 4). At this particular site (Table 2), the targets are TC photoproducts [35,52]. These lesions could be (6-4) photoproducts or CPDs, but the former is a strong obstacle to be replicated by Pol␩ in vivo [53]. The raise of this type of mutations in the strain overproducing AtPOLH may be explained considering that the target lesion of this type of mutant is mainly TC cyclobutane pyrimidine dimer. The cytosine base in DNA undergoes hydrolytic deamination at an elevated rate when it is included in a CPD. Takasawa et al. [54] observed that in vitro, human Pol␩ bypassed with the same efficiency TT and TU cis–syn cyclobutane dimers, incorporating two adenylates and concluded that in TC dimers, C is deaminated to U and the incorporation of dAMP opposite the U component induces C to T transitions. On the other hand, PolV predominantly introduce dGMP opposite the 3 C or 3 U of the TC or TU dimers, and therefore it is not expected to produce mutations at this site. Finally considering the number of tyrosine tRNA ochre suppressor mutations arising after UV irradiation, it is concluded that UV light does not produce this class of mutants (Fig. 5). This result could be considered as a negative control, since the DNA sequence change requisite to generate this particular mutation would be a transversion at a cytosine between two adenines or at a guanine between two thymines (Table 2), places where pyrimidine–pyrimidine photoproducts could not be formed. Fig. 5 also shows a decrease of this type of mutant, when bacteria are UV irradiated. This antimutagenic outcome has been observed previously [55]. It was suggested that tyrosine tRNA ochre suppressor mutations probably occurred at apurinic sites, and that some elements of the machinery involved in the mechanism

Fig. 5. Spontaneous and UV yields for tyrosine tRNA ochre suppressor mutations in E. coli IC1141 strain transformed with pMAL (pMAL dT) or transformed with pMAL-AtPOLH (pMAL eta dT).

of spontaneous mutagenesis are engaged processing UV DNA damages [55]. 4. Discussion This paper describes the identification of AtPolH from A. thaliana, a Y-family DNA polymerase homolog to Pol␩ from yeast, human and other organisms. The DNA polymerase ␩ was the second eukaryotic polymerase found to be implicated in DNA translesion synthesis

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[12,20,50,56]. We observed, as expected, that AtPOLH complements the UV sensitivity phenotype of a yeast strain deficient in DNA polymerase ␩. The AtPOLH gene has an ubiquitous expression, suggesting a fundamental function for this enzyme as a tolerance mechanism to neutralize DNA damages. This pattern of expression would not be predicted for a gene encoding a polymerase involved only in specific bypass of UV-lesions as cis–syn dipyrimidine photoproducts, since some tissues are not exposed to light, as is the case of roots. Therefore, the expression of this gene in all tissues, supports the idea that this polymerase could carry out TLS of other lesions originated in the DNA by chemical or physical agents, for example endogenous reactive oxygen free radicals. The analysis of arg+ prototrophic mutants with defective T4 phages showed that when AtPOLH is overexpressed in E. coli, there is a change in the type of UVinduced mutations. In E. coli, the capacity of pyrimidine dimers to block DNA replication in vivo was established with cis–syn thymine–thymine cyclobutane dimers in a single-stranded M13 viral DNA. In this system it was found that replication stops at least in 99.5% of the cases [57]. However, if the SOS response was induced, pyrimidine dimers did not block replication, because of the induction of umuC and umuD genes encoding DNA polymerase V, which is proficient at bypassing UV-induced pyrimidine dimers [10]. The in vivo spectrum of SOSinduced mutants shows preference of T to C transitions in thymine dimers [58,59] and correlates with the bypass specificity of DNA polymerase V in vitro, which usually introduces a G opposite a T included in the dimer [60]. The expression of AtPOLH produces a decrease in the number of back mutants and an increase of specific tRNA ochre suppressor mutations. This observation suggests that AtPOLH might compete with bacterial DNA polymerase V in TLS and that TT or TC cyclobutane pyrimidine dimers are bypassed by Arabidopsis Pol␩ incorporating two adenylates. To test this hypothesis, we have carried out experiments in UmuC minus background. Contrary to our expectative this mutant continued being “unmutable” by UV light and there was not an increase of specific suppressors (data not shown). We think that this result may be due to the presence of RecA, that strongly bind to damaged DNA, and in the absence of PolV remains bind [61,62]. In this scenario, AtPOLH could not have access to the damage. It has been shown that (6-4) TT lesion, which distorts the DNA helix to a much greater extent than a cis–syn TT dimer, cannot be bypassed by Pol␩ in vitro [18]. Replication through (6-4) TT lesions has been studied in vivo in S. cerevisiae wild type and rad30 strains. It was found

that in the wild type strain, (6-4) TT lesions were replicated both inaccurately and accurately 60% and 40% of the time, respectively, while in the rad30 mutant the level of bypass remained almost 100% error-free [63]. These results indicate that Pol␩ is error-prone in vivo for this photoproduct. On the other hand, it was shown in vitro that Pol␩ incorporates a G opposite the 3 T of a (6-4) TT lesion [18], which should originate a T to C substitution [64]. Because (6-4) photoproducts are formed more frequently at 5 -TC-3 that at TT sites, it has been suggested [7,65] that Pol␩ contributes also to the errorfree bypass of (6-4) lesions formed at TC, given that the incorporation of a G opposite the 3 C of TC photoproducts, followed by extension for DNA polymerase ␨, may provide the error-free bypass at these sites. Contrary to expectation, the results of Fig. 4 show an increase of specific ochre mutations in bacteria expressing AtPOLH (Table 2). Other authors have reported that supF UVinduced mutation frequency increased up to 3.6-fold in the siRNA Pol␩ knockdown human cells, confirming the importance of this polymerase in UV mutation protection [66]. However, almost 90% of the mutations were C to T transitions, both in control and in the siRNA knockdown cells. The authors postulate that this kind of mutation could be explained by cytosine deamination in dimers. These data support the idea that C to T transitions observed in Fig. 4 might also be a result of cytosine deamination. Sliding clamp proteins are found in all organisms and are called proliferating cell nuclear antigen (PCNA) in eukaryotes and beta clamp in prokaryotes. Both PCNA and beta clamp form a ring around DNA. The sliding clamp slides along DNA, enabling rapid and processive DNA replication by polymerase [67]. The beta-clamp binding motif of TLS polymerases of E. coli is QLxLF, similar to QxxLxxFF, which is the conserved PCNA binding motif of Pol␩ and other proteins [47]. Therefore, there is a possibility that AtPOLH could participate displacing PolV in vivo. In addition, since several DNA polymerases are involved in lesion bypass interacting with PCNA or beta clamp, it is likely that the bypass mechanism of TLS polymerases is evolutionary conserved [68]. Given that there is relatively little information on how Pol␩ gains access at the lesion site and exits from it, bacteria could be used as an in vivo tool for studying some features of the mechanism of eukaryotic TLS. Acknowledgements We are grateful to Dr. C. de la Hera for critical reading of the manuscript and Dr. R.R. Ariza, V. Montiel and I. Caballero for their help. We also appreciate

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the generous gift of yeast strains to Dr. E. Heidenreich. M.J. Santiago was supported by a fellowship of Programa Nacional de Formaci´on de Profesorado Universitario del Ministerio de Educaci´on y Ciencia. This work was financed by grants from Ministerio de Ciencia y Tecnolog´ıa (BOS2000-0894 and BMC200304350) and from the Junta de Andaluc´ıa (CVI-272). This research has been carried out in agreement with current laws governing genetic experimentation in Spain.

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