Translesion synthesis DNA polymerases η, ι, and ν promote mutagenic replication through the anticancer nucleoside cytarabine

Cytarabine (AraC) is the mainstay for the treatment of acute myeloid leukemia. Although complete remission is observed in a large proportion of patients, relapse occurs in almost all the cases. The chemotherapeutic action of AraC derives from its ability to inhibit DNA synthesis by the replicative polymerases (Pols); the replicative Pols can insert AraCTP at the 3′ terminus of the nascent DNA strand, but they are blocked at extending synthesis from AraC. By extending synthesis from the 3′-terminal AraC and by replicating through AraC that becomes incorporated into DNA, translesion synthesis (TLS) DNA Pols could reduce the effectiveness of AraC in chemotherapy. Here we identify the TLS Pols required for replicating through the AraC templating residue and determine their error-proneness. We provide evidence that TLS makes a consequential contribution to the replication of AraC-damaged DNA; that TLS through AraC is conducted by three different pathways dependent upon Polη, Polι, and Polν, respectively; and that TLS by all these Pols incurs considerable mutagenesis. The prominent role of TLS in promoting proficient and mutagenic replication through AraC suggests that TLS inhibition in acute myeloid leukemia patients would increase the effectiveness of AraC chemotherapy; and by reducing mutation formation, TLS inhibition may dampen the emergence of drug-resistant tumors and thereby the high incidence of relapse in AraC-treated patients.

Acute myeloid leukemia (AML) 3 is a cancer of the myeloid line of blood cells. In AML, myeloblast, an immature precursor of white blood cells in normal hematopoiesis, undergoes genetic changes that prevent its normal differentiation. The undifferentiated immature clone of myeloblasts continues to proliferate, jeopardizing the production of normal white blood cells. The replacement of normal blood cells with leukemia cells in bone marrow causes a large reduction not only in white blood cells but also in platelets and red blood cells. As a result, AML patients suffer from an increased risk of infection, anemia, and bleeding (1). The incidence of AML increases with age, and it accounts for ϳ90% of acute leukemias in adults. Cytarabine (1-␤-D-arabinofuranosyl cytosine) (AraC) is a drug of choice in AML treatment (2)(3)(4). Although complete remission is observed in 50 -75% of cases, relapse occurs in almost all of these cases (5,6), requiring further treatment that may include postremission chemotherapy, stem cells transplantation, or immunotherapy.
AraC differs from 2Ј-deoxycytidine by the presence of an additional hydroxyl group at the C2Ј position of the 2Ј-deoxyribose, and AraC differs from cytidine in that the 2Ј-OH of the arabinose sugar points in a direction opposite from that of the 2Ј-OH of the ribose sugar in ribonucleotides (Fig. 1A). The chemotherapeutic action of AraC derives from its ability to inhibit DNA replication. Inside the cell, AraC is converted to a triphosphate (7), and AraCTP competes with dCTP for incorporation into DNA. The replicative polymerases (Pols) can insert AraC into DNA, but they are inhibited at extending from AraC at the 3Ј terminus (8 -10). In the next replication cycle, the presence of AraC in the template strand will be further inhibitory to DNA replication. However, human cells harbor a number of translesion synthesis (TLS) DNA Pols that can, in principle, surmount the chemotherapeutic action of AraC both by extending DNA synthesis from AraC terminated 3Ј ends and by replicating through AraC in the template strand. As such, TLS Pols may play a pre-eminent role in reducing the chemotherapeutic impact of AraC, but there exists little information on how the TLS Pols enhance the replication potential of AraC-treated cells.
Biochemical and structural studies have indicated that different TLS Pols are adapted for replicating through different types of DNA lesions, and depending upon the DNA lesion, a particular TLS Pol may carry out only the insertion or the extension step of TLS, or it could perform both the steps of TLS (11). For example, among the Y-family Pols, Pol has the unique ability to accommodate two template residues in its active site, and thus it can efficiently replicate through UV-induced cyclobutane pyrimidine dimers (12)(13)(14)(15). The ability of Pol to push the template purine A or G residue into a syn conformation and to form an Hoogsteen bp with the incoming T or C, respectively, enables it to insert nucleotides (nts) opposite DNA lesions that impair Watson-Crick base pairing (16 -18). Rev1 pushes the templating G residue into a solvent-filled cavity, and an Arg residue in Rev1 forms hydrogen bonds with the This work was supported by National Institutes of Health Grant CA200575.
The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1  cro ARTICLE incoming C (19,20); this protein template-directed mechanism of nt incorporation allows Rev1 to insert a C opposite N 2 -dG adducts that protrude into the DNA minor groove (21). Pol is highly adapted for extending from such minor groove DNA lesions (22), whereas Pol, a member of the B-family of Pols, can extend synthesis from the nt opposite from a large variety of DNA lesions (11,17,(23)(24)(25).
Although we now have considerable information on how TLS Pols can replicate through different kinds of DNA lesions generated from exogenous and endogenous sources, this information pertains largely to DNA lesions that disrupt the base moiety and not the sugar moiety. In the absence of genetic and cellular studies, there is no clear understanding of the relevance of TLS in AraC-treated cells, and the identity of TLS Pols involved in AraC bypass is also unknown. As such, the role of TLS Pols in reducing the chemotherapeutic potential of AraC in AML remains undetermined. The mutagenic potential of TLS through AraC is also unknown. There is no information available on which TLS Pols function in an error-free manner and which function in a mutagenic fashion. Because error-prone replication by TLS Pols through the AraC lesion may contribute to the emergence of drug-resistant tumors and to the relapse of cancers, knowledge of which TLS Pols function in an error-prone manner could suggest means for preventing the emergence of drug-resistant tumors and for reducing refractory AML.
Here we identify the TLS Pols required for replicating through AraC in human cells. Our data indicate that TLS through AraC occurs via three different pathways dependent upon Pol, Pol, and Pol, respectively. TLS through AraC generates a considerable level of mutagenesis as ϳ10% of TLS products harbor mutations in which an A is inserted opposite AraC, and all these Pols contribute to mutagenic TLS. The rather high mutagenicity of AraC may contribute to the emergence of drug-resistant tumors and to the relapse of cancers.

Genetic control of TLS through AraC in human cells
AraC (Fig. 1A) was incorporated in the lacZ target sequence in the leading strand in the SV40-based duplex plasmid system in which bidirectional replication initiates from a replication origin (Fig. 1B). Because the lacZ sequence in the AraC-containing strand is in frame, both error-free and error-prone TLS mechanisms generate blue colonies. The other DNA strand lacking AraC contains a ϩ1 frameshift; hence, the lacZ gene in this strand is nonfunctional. The two DNA strands are further distinguished by the kanϩ (kanamycin resistance) gene in the AraC-containing strand and the kanϪ gene in the other strand (Fig. 1B). In this plasmid system, the number of blue colonies among the total colonies that grow on LB ϩ Kan plates gives a very reliable estimate of TLS frequency (26).
To identify the TLS Pols involved in replicating through AraC, we examined the effects of siRNA depletions on TLS frequency. In normal human fibroblasts (HFs) treated with control (NC) siRNA, TLS occurred with a frequency of ϳ47%, and depletion of Pol or the Rev3 catalytic subunit of Pol had no effect on TLS frequency ( Table 1). Depletion of Pol or Pol, however, reduced TLS frequency to ϳ33%, and depletion of Pol conferred a reduction to ϳ27% (Table 1). To determine whether Pols , , and carry out TLS independently or

TLS through anticancer nucleoside cytarabine
whether any two of these Pols function together in one TLS pathway, in which one Pol would insert a nt opposite AraC and the other Pol would extend synthesis from the inserted nt, we examined the effects of co-depletion of any two of these Pols on TLS frequency. Co-depletion of Pol and Pol reduced TLS frequency to ϳ23%, and co-depletion of Pol with Pol reduced TLS frequency to ϳ19% ( Table 1). The observation that co-depletion of Pol with Pol or with Pol conferred a greater reduction in TLS frequency than that seen upon their individual depletion suggested that Pol functions independently of Pol or Pol. The result that the co-depletion of Pol and Pol reduced TLS frequency to ϳ14% indicated that Pol and Pol function independently of one another (Table 1). Altogether, these results indicated that TLS through AraC is mediated via three independent pathways dependent upon Pol, Pol, and Pol, respectively.
To verify this conclusion, we examined the effects of depletion of Pol or Pol in XPV cells defective in Pol. In XPV cells, TLS through AraC occurs at a frequency of ϳ37%, and it is reduced to ϳ25% in Pol-depleted cells and to ϳ21% in Poldepleted cells (Table 2). This reduction in TLS frequency confirms the inference that Pol and Pol act independently of Pol. The observation that co-depletion of Pol and Pol in XPV cells reduced TLS frequency to ϳ7% (Table 2) adds further support to the conclusion that Pols , , and function independently of one another in mediating TLS through AraC.

Mutagenicity of TLS opposite AraC
In HFs treated with control siRNA, TLS through AraC incurs significantly elevated mutagenicity, because ϳ9% of TLS products harbor a mutational change in which an A is incorporated opposite AraC (Table 3). Depletion of Pol or Pol reduced the frequency of mutagenic TLS to ϳ7.5%, and depletion of Pol increased the frequency of mutagenic TLS to ϳ14%. Similar to that in WT HFs treated with control siRNA, in HFs depleted for Pol, Pol, or Pol, mutagenic TLS occurred via an A incorporation (Table 3). Even though all these Pols conduct errorprone TLS opposite AraC, the greater increase in mutation frequency in Pol-depleted cells than in Pol-or Pol-depleted cells suggests that Pol promotes a less error-prone mode of TLS opposite AraC than Pol or Pol.

Replication through AraC by purified TLS Pols
Our genetic evidence in human cells that TLS through AraC is mediated by Pol-, Pol-, and Pol-dependent pathways and that all these Pols contribute to mutagenic TLS by inserting an A opposite AraC suggested that in biochemical assays these Pols will insert predominantly a G and less frequently an A opposite AraC. To assess this, we examined their ability for replicating through AraC in the presence of dATP, dTTP, dGTP, or dCTP, or all four dNTPs (Fig. 2). Opposite AraC, Pol inserts a G but it also inserts an A, T, or C; T or C, however, are inserted less well than an A ( Fig. 2A). Opposite undamaged C, Pol exhibits high error-proneness, misincorporating all the nucleotides. In the presence of all four dNTPs, Pol replicates through AraC proficiently ( Fig. 2A). In contrast to Pol, Pol inserts only a G opposite AraC, and it is inhibited at extending synthesis any further ( Fig. 2A). Pol also inserts only a G opposite AraC, and in the presence of all four dNTPs, it inserts a G and then extends synthesis (Fig. 2B).

TLS Pols promote survival of AraC-treated cells
To determine the contribution of Pol, Pol, and Pol to survival in AraC-treated cells, we incubated WT HFs depleted for TLS Pols in medium containing 30 M AraC for 48 h. Compared with cells treated with NC siRNA, survival was reduced to ϳ75% in cells depleted for Pol, Pol, or Pol, and co-depletion of Pol with Pol or Pol or co-depletion of Pol with Pol reduced survival to ϳ55% (Fig. 3A). In AraC-treated XPV HFs, depletion of Pol or Pol reduced survival, and co-depletion of Pol with Pol caused a further reduction in survival (Fig. 3B). Overall, all three Pols contribute approximately equally to the survival of AraC-treated cells.

Genetic pathways for replicating through AraC in human cells
From siRNA depletion of TLS Pols in normal HFs, we inferred the involvement of Pols , , and in TLS through AraC, and based on co-depletion analyses in HFs and XPV cells, we concluded that these Pols act independently in replicating through AraC (Fig. 4). The proficient ability of purified Pol to

TLS through anticancer nucleoside cytarabine
insert a nt opposite AraC, and to extend synthesis from the inserted nt suggests that Pol alone could conduct TLS through AraC. By contrast to Pol, Pol inserts a nt opposite AraC, but it is very inefficient in extending synthesis from the inserted nt, suggesting that the extension step is performed by an as-yetunidentified Pol. The proficient ability of Pol for inserting a nt opposite AraC and for extending synthesis suggests that Pol alone could replicate through AraC in human cells (Fig. 4).

High error-proneness of TLS opposite AraC
In normal human cells, Pols , , and carry out highly error-prone TLS opposite AraC. Although the high errorproneness of Pol for TLS in human cells conforms with the error-prone synthesis by purified Pol opposite AraC, the error-proneness of TLS by Pols and in human cells is incongruent with the error-free TLS performed by these   (26 -34) occurs with a much higher fidelity than indicated from the fidelity of purified Pols. The much higher fidelity of TLS Pols in human cells than that indicated from in vitro biochemical analyses can be rationalized by assuming that TLS Pols in human cells are components of multiprotein ensembles, and the fidelity of TLS Pols in these ensembles is actively modulated by protein-protein interactions and post-transcriptional modifications.
Contrawise, the acquisition of reduced fidelity by Pol and Pol in TLS opposite AraC in human cells would suggest that protein-protein interactions and post-transcriptional modifications contribute to reducing the fidelity of TLS Pols opposite AraC rather than to its enhancement. The striking divergence in the fidelity of TLS Pols opposite AraC versus opposite other DNA lesions in human cells may accrue from the fact that all the other DNA lesions for which we have analyzed the genetic control and fidelity of TLS thus far are generated from normal cellular reactions or from prevalent environmental sources; consequently, strong selection pressure for maintaining cellular homeostasis would have led to the acquisition of predominantly error-free TLS mechanisms. Because AraC is not a byproduct of cellular reactions or generated from persistent environmental exposure, TLS mechanisms opposite this chemotherapeutic drug would have been under no selection pressure to adapt to a more error-free mode; instead, the mechanisms that have evolved to adapt TLS Pols to act in a more error-free manner opposite the various more prevalent DNA lesions could have caused TLS Pols to operate opposite AraC in a more error-prone manner in human cells than in the purified Pol.

Role of TLS in countering the chemotherapeutic potential of AraC
TLS Pols will impact the chemotherapeutic potential of AraC first by extending synthesis from AraCMP at the 3Ј terminus of the nascent DNA strand and second by replicating through AraC incorporated in the template strand. Biochemical studies have indicated a role of Pol in extending synthesis from AraC at the 3Ј terminus, and structural studies have shown that Pol can accommodate AraC via specific hydrogen bonding and stacking interactions (35). Our results that TLS Pols , , and can promote replication through AraC and that their inactivation reduces survival of AraC-treated cells suggest that TLS through AraC would contribute to reducing the effectiveness of AraC chemotherapy. In addition, our evidence that TLS through AraC generates a considerably high level of mutations explains the high mutagenicity conferred by AraC treatment (36 -40) and suggests that TLS-induced mutagenicity would contribute to the emergence of drug-resistant tumors and to the relapse of cancers.

Construction of plasmid vectors containing an AraC and TLS assays
The 16-mer oligonucleotides containing an AraC were purchased from Trilink Biotechnologies, and the in-frame target sequence of the lacZЈ gene in the resulting vector is shown in Fig. 1B. The WT kanamycin gene was placed on the DNA strand containing AraC, and in this DNA strand laczЈ is inframe and functional for ␤-galactosidase. The opposite DNA strand harbors an SpeI restriction site containing a ϩ1 frameshift, which makes it nonfunctional for ␤-galactosidase. Details of TLS assays have been published before (26,32).

Translesion synthesis assays in human cells and mutational analyses of TLS products
WT (GM637) or XPV (XP30RO) HFs were grown in Dulbecco's modified Eagle's medium (GeneDepot) containing high D-glucose (4500 mg/liter), Phenol red (15 mg/liter), and sodium pyruvate (110 mg/liter) with 10% fetal bovine serum (GeneDepot) and plated in 6-well plates at 70% confluence (approximately, 3 ϫ 10 5 cells/well). The cells were transfected with 100 pmol of siRNAs with Lipofectamine 2000 (Invitrogen). For the simultaneous siRNA knockdown of two genes, 100 pmol of siRNAs for each gene were mixed and transfected. After 48 h of incubation, the heteroduplex target vector DNA (1 g) and 50 pmol of siRNA (second transfection) were co-transfected with Lipofectamine 2000 (Invitrogen). After 30 h of incubation, plasmid DNA was rescued from the cells by the alkaline lysis method and digested with DpnI to remove unreplicated plasmid DNA. The plasmid DNA was then transformed into Escherichia coli XL1-Blue super competent cells (Stratagene). Transformed bacterial cells were diluted in 1 ml of SOC medium and plated on LB/kan (25 g/ml kanamycin) (Sigma). TLS analyses and mutational analyses of TLS products were carried out as described previously (26).

TLS through anticancer nucleoside cytarabine siRNA sequences used for knockdowns of human TLS Pols
The siRNA sequences used for knockdowns of Pol, Pol, and Rev3 have been published previously (26). The siRNA sequence used for Pol depletion is 5Ј-CCCAAUUCAGAUU-ACUACATT-3Ј.

AraC survival assay
WT (GM637) or XPV (XP30RO) HFs were transfected with siRNA, and 48 h after siRNA transfection, the cells were incubated with 30 M AraC (Sigma) in fresh growth medium for 48 h. AraC cytotoxicity was determined by MTS assay (Promega). Briefly, 100 l of MTS assay solutions were added to each well containing cells and incubated for 30 min. Cell viability was determined by measuring OD at 490 nM. Four independent experiments were carried out.

Protein expression and purification
Full-length human Pol and Pol were expressed as GSTtagged fusion proteins from plasmids pR30.186 and pPOL114, respectively, and purified from yeast cells as described (41)(42)(43). The GST tags were cleaved from each DNA Pol by treatment with prescission protease, leaving a 7-amino acid linker peptide at the N terminus of each protein. To express human Pol lacking the proline-rich C-terminal 39 residues (44) in yeast, a 2.6-kb E. coli codon optimized cDNA encoding amino acids 1-861 of the 900-amino acid protein was synthesized. The cDNA was amplified by PCR to add flanking 5Ј and 3Ј BamHI restriction endonuclease sites, and the fragment was cloned in frame with a FLAG metal affinity tag-SUMO* tag under control of a galactose inducible phosphoglycerate kinase promoter in plasmid pPM1514, generating plasmid pBJ2086. SUMO* contains residues 1-98 of the yeast SMT3 encoded protein and harbors mutations R64T and R71E, rendering it resistant to cellular sumo-proteases. Yeast strain YRP654 was transformed with plasmid pBJ2086, and cells were grown as described (41). Protein expression was induced by the addition of 2% galactose, and cells were grown for 16 h. Pol(1-861) was affinity-purified using anti-FLAG agarose. Clarified protein extract was prepared from 10 g of yeast cells disrupted by French press as described (41), except that breakage buffer additionally contained 0.1% Triton X-100. The clarified protein extract was rocked with 0.1 ml of M2 ␣FLAG-agarose (Sigma) for 3 h and subsequently washed with 20 volumes of 1ϫ GBB (GST binding buffer) containing 500 mM NaCl and 0.1% Triton X-100. The M2 ␣FLAG-agarose beads were equilibrated in 1ϫ GBB (GST binding buffer) containing 150 mM NaCl and 0.01% Triton X-100, and protein was eluted by the addition of 0.1 mg/ml FLAG peptide. Eluted protein was aliquoted and frozen at Ϫ70°C.

DNA polymerase assays
DNA substrates consisted of a radiolabeled oligonucleotide primer annealed to a 75-nt-oligonucleotide DNA template by heating a mixture of primer/template at a 1:1.5 molar ratio to 95°C and allowing it to cool to room temperature for several hours. The template 75-mer oligonucleotide contained the sequence 5Ј-AGC AAG TCA CCA ATG TCT AAG AGT TCG TAT CAT GCC TAC ACT GGA GTA CCG GAG CAT CGT CGT GAC TGG GAA AAC-3Ј, and it harbored an undamaged C or an AraC at the underlined position. For examining the incorporation of dATP, dTTP, dCTP, or dGTP nucleotides, or of all four dNTPs, a 44-mer primer 5Ј-GTT TTC CCA GTC ACG ACG ATG CTC CGG TAC TCC AGT GTA GGC AT-3Ј was annealed to the above mentioned 75-mer templates.
The standard DNA polymerase reaction (5 l) contained 25 mM Tris⅐HCl (pH 7.5), 5 mM MgCl 2 , 1 mM dithiothreitol, 100 g/ml BSA, 10% glycerol, 10 nM DNA substrate, and 1 nM of Pol, Pol, or Pol. For nucleotide incorporation assays with Pol or Pol, 25 M dATP, dTTP, dCTP, or dGTP (Roche Biochemicals) were used, and for examining synthesis through the undamaged C or AraC, all four dNTPs (25 M each) were used. For nucleotide incorporation assays with Pol, 10 M dATP, dTTP, dCTP, or dGTP were used, and for examining synthesis through the undamaged C or AraC, all four dNTPs (10 M each) were used. The reactions were carried out for 10 min at 37°C. Reaction products were resolved on a 12% polyacrylamide gel containing 8 M urea and analyzed by a PhosphorImager.