Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.

The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3-/-p53-/- mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1-/- and Polh-/-Poli-/-Polk-/- MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.

Hypersensitivity of mouse embryonic fibroblast cells defective for DNA polymerases η, ι and κ to various genotoxic compounds: Its potential for application in chemical genotoxic screening.

Genotoxic agents cause modifications of genomic DNA, such as alkylation, oxidation, bulky adduct formation, and strand breaks, which potentially induce mutations and changes to the structure or number of genes. Majority of point mutations are generated during error-prone bypass of modified nucleotides (translesion DNA synthesis, TLS); however, when TLS fails, replication forks stalled at lesions eventually result in more lethal effects, formation of double-stranded breaks (DSBs). Here we compared sensitivities to various compounds among mouse embryonic fibroblasts derived from wild-type and knock-out mice lacking one of the three Y-family TLS DNA polymerases (Polη, Polι, and Polκ) or all of them (TKO). The compounds tested in this study include genotoxins such as methyl methanesulfonate (MMS) and nongenotoxins such as ammonium chloride. We found that TKO cells exhibited the highest sensitivities to most of the tested genotoxins, but not to the non-genotoxins. In order to quantitatively evaluate the hypersensitivity of TKO cells to different chemicals, we calculated ratios of half-maximal inhibitory concentration for WT and TKO cells. The ratios for 9 out of 10 genotoxins ranged from 2.29 to 5.73, while those for 5 nongenotoxins ranged from 0.81 to 1.63. Additionally, the two markers for DNA damage, ubiquitylated proliferating cell nuclear antigen and γ-H2AX after MMS treatment, were accumulated in TKO cells more greatly than in WT cells. Furthermore, following MMS treatment, TKO cells exhibited increased frequency of sister chromatid exchange compared with WT cells. These results indicated that the hypersensitivity of TKO cells to genotoxins resulted from replication fork stalling and subsequent DNA double-strand breaks, thus demonstrating that TKO cells should be useful for evaluating chemical genotoxicity.

Somatic hypermutation of immunoglobulin genes in Rad18 knockout mice.

Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is triggered by the activity of activation-induced cytidine deaminase (AID). AID induces DNA lesions in variable regions of Ig genes, and error-prone DNA repair mechanisms initiated in response to these lesions introduce the mutations that characterize SHM. Error-prone DNA repair in SHM is proposed to be mediated by low-fidelity DNA polymerases such as those that mediate trans-lesion synthesis (TLS); however, the mechanism by which these enzymes are recruited to AID-induced lesions remains unclear. Proliferating cell nuclear antigen (PCNA), the sliding clamp for multiple DNA polymerases, undergoes Rad6/Rad18-dependent ubiquitination in response to DNA damage. Ubiquitinated PCNA promotes the replacement of the replicative DNA polymerase stalled at the site of a DNA lesion with a TLS polymerase. To examine the potential role of Rad18-dependent PCNA ubiquitination in SHM, we analyzed Ig gene mutations in Rad18 knockout (KO) mice immunized with T cell-dependent antigens. We found that SHM in Rad18 KO mice was similar to wild-type mice, suggesting that Rad18 is dispensable for SHM. However, residual levels of ubiquitinated PCNA were observed in Rad18 KO cells, indicating that Rad18-independent PCNA ubiquitination might play a role in SHM.

Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases.

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.

Different sets of translesion synthesis DNA polymerases protect from genome instability induced by distinct food-derived genotoxins.

DNA lesions, induced by genotoxic compounds, block the processive replication fork but can be bypassed by specialized translesion synthesis (TLS) DNA polymerases (Pols). TLS safeguards the completion of replication, albeit at the expense of nucleotide substitution mutations. We studied the in vivo role of individual TLS Pols in cellular responses to benzo[a]pyrene diolepoxide (BPDE), a polycyclic aromatic hydrocarbon, and 4-hydroxynonenal (4-HNE), a product of lipid peroxidation. To this aim, we used mouse embryonic fibroblasts with targeted disruptions in the TLS-associated Pols η, ι, κ, and Rev1 as well as in Rev3, the catalytic subunit of TLS Polζ. After exposure, cellular survival, replication fork progression, DNA damage responses (DDR), and the induction of micronuclei were investigated. The results demonstrate that Rev1, Rev3, and, to a lesser extent, Polη are involved in TLS and the prevention of DDR and of DNA breaks, in response to both agents. Conversely, Polκ and the N-terminal BRCT domain of Rev1 are specifically involved in TLS of BPDE-induced DNA damage. We furthermore describe a novel role of Polι in TLS of 4-HNE-induced DNA damage in vivo. We hypothesize that different sets of TLS polymerases act on structurally different genotoxic DNA lesions in vivo, thereby suppressing genomic instability associated with cancer. Our experimental approach may provide a significant contribution in delineating the molecular bases of the genotoxicity in vivo of different classes of DNA-damaging agents.

The vital role of polymerase ζ and REV1 in mutagenic, but not correct, DNA synthesis across benzo[a]pyrene-dG and recruitment of polymerase ζ by REV1 to replication-stalled site.

The DNA synthesis across DNA lesions, termed translesion synthesis (TLS), is a complex process influenced by various factors. To investigate this process in mammalian cells, we examined TLS across a benzo[a]pyrene dihydrodiol epoxide-derived dG adduct (BPDE-dG) using a plasmid bearing a single BPDE-dG and genetically engineered mouse embryonic fibroblasts (MEFs). In wild-type MEFs, TLS was extremely miscoding (>90%) with G → T transversions being predominant. Knockout of the Rev1 gene decreased both the TLS efficiency and the miscoding frequency. Knockout of the Rev3L gene, coding for the catalytic subunit of pol ζ, caused even greater decreases in these two TLS parameters; almost all residual TLS were error-free. Thus, REV1 and pol ζ are critical to mutagenic, but not accurate, TLS across BPDE-dG. The introduction of human REV1 cDNA into Rev1(-/-) MEFs restored the mutagenic TLS, but a REV1 mutant lacking the C terminus did not. Yeast and mammalian three-hybrid assays revealed that the REV7 subunit of pol ζ mediated the interaction between REV3 and the REV1 C terminus. These results support the hypothesis that REV1 recruits pol ζ through the interaction with REV7. Our results also predict the existence of a minor REV1-independent pol ζ recruitment pathway. Finally, although mutagenic TLS across BPDE-dG largely depends on RAD18, experiments using Polk(-/-) Polh(-/-) Poli(-/-) triple-gene knockout MEFs unexpectedly revealed that another polymerase(s) could insert a nucleotide opposite BPDE-dG. This indicates that a non-Y family polymerase(s) can insert a nucleotide opposite BPDE-dG, but the subsequent extension from miscoding termini depends on REV1-polζ in a RAD18-dependent manner.

Overlapping in short motif sequences for binding to human REV7 and MAD2 proteins.

Polζ, a DNA polymerase specialized for translesion DNA synthesis (TLS), is comprised of two subunits, the REV3 catalytic subunit and the REV7 accessory subunit. The human REV7 (hREV7) protein is known to interact with hREV3, hREV1 (another TLS protein) and some other proteins such as ADAM9 (a disintegrin and metalloprotease) and ELK-1 (an Ets-like transcription factor). hREV7 is alternatively termed hMAD2L2, because its primary sequence shows 26% identity to that of hMAD2 that plays crucial roles in spindle assembly checkpoint (SAC) via interactions with hMAD1 or hCDC20. Here, we have investigated the molecular basis for the interactions of hREV7/MAD2L2 and hMAD2 with their binding partners. Our results showed that a short sequence of hREV3 is necessary and sufficient for interaction with hREV7. Surprisingly, hMAD2 also binds to the hREV7-binding sequence in hREV3, whereas hMAD2 does not bind to a similar sequence in ADAM9 or ELK-1 and hREV7 does not bind to the hMAD2-binding sequence in hMAD1 or hCDC20. We discuss how hREV7 and hMAD2 recognize their binding partners, and how hREV3 and hREV7 might be involved in SAC.

Structural basis for novel interactions between human translesion synthesis polymerases and proliferating cell nuclear antigen.

Translesion synthesis (TLS) is a DNA damage tolerance mechanism that allows continued DNA synthesis, even in the presence of damaged DNA templates. Mammals have multiple DNA polymerases specialized for TLS, including Poleta, Poliota, and Polkappa. These enzymes show preferential bypass for different lesions. Proliferating cell nuclear antigen (PCNA), which functions as a sliding clamp for the replicative polymerase Poldelta, also interacts with the three TLS polymerases. Although many PCNA-binding proteins have a highly conserved sequence termed the PCNA-interacting protein box (PIP-box), Poleta, Poliota, and Polkappa have a noncanonical PIP-box sequence. In response to DNA damage, Lys-164 of PCNA undergoes ubiquitination by the RAD6-RAD18 complex, and the ubiquitination is considered to facilitate TLS. Consistent with this, these three TLS polymerases have one or two ubiquitin binding domains and are recruited to replication forks via interactions with ubiquitinated PCNA involving the noncanonical PIP-box and ubiquitin binding domain. However, it is unclear how these TLS polymerases interact with PCNA. To address the structural basis for interactions between different TLS polymerases and PCNA, we determined crystal structures of PCNA bound to peptides containing the noncanonical PIP-box of these polymerases. We show that the three PIP-box peptides interact with PCNA in different ways, both from one another and from canonical PIP-box peptides. Especially, the PIP-box of Poliota adopts a novel structure. Furthermore, these structures enable us to speculate how these TLS polymerases interact with Lys-164-monoubiquitinated PCNA. Our results will provide clues to understanding the mechanism of preferential recruitment of TLS polymerases to the stalled forks.

Penicilliols A and B, novel inhibitors specific to mammalian Y-family DNA polymerases.

Penicilliols A (1) and B (2) are novel 5-methoxy-3(2H)-furanones isolated from cultures of a fungus (Penicillium daleae K.M. Zalessky) derived from a sea moss, and their structures were determined by spectroscopic analyses. These compounds selectively inhibited activities of eukaryotic Y-family DNA polymerases (pols) (i.e., pols eta, iota and kappa), and compound 1 was a stronger inhibitor than compound 2. Among mammalian Y-family pols, mouse pol iota activity was most strongly inhibited by compounds 1 and 2, with IC(50) values of 19.8 and 32.5 microM, respectively. On the other hand, activities of many other pols, such as A-family (i.e., pol gamma), B-family (i.e., pols alpha, delta and epsilon) or X-family (i.e., pols beta, lambda and terminal deoxynucleotidyl transferase), and some DNA metabolic enzymes, such as calf primase of pol alpha, human immunodeficiency virus type-1 (HIV-1) reverse transcriptase, human telomerase, T7 RNA polymerase, mouse IMP dehydrogenase (type II), human topoisomerases I and II, T4 polynucleotide kinase or bovine deoxyribonuclease I, are not influenced by these compounds. In conclusion, this is the first report on potent inhibitors of mammalian Y-family pols.

Interaction with DNA polymerase eta is required for nuclear accumulation of REV1 and suppression of spontaneous mutations in human cells.

Defects in the gene encoding human Poleta result in xeroderma pigmentosum variant (XP-V), an inherited cancer-prone syndrome. Poleta catalyzes efficient and accurate translesion DNA synthesis (TLS) past UV-induced lesions. In addition to Poleta, human cells have multiple TLS polymerases such as Poliota, Polkappa, Polzeta and REV1. REV1 physically interacts with other TLS polymerases, but the physiological relevance of the interaction remains unclear. Here we developed an antibody that detects the endogenous REV1 protein and found that human cells contain about 60,000 of REV1 molecules per cell as well as Poleta. In un-irradiated cells, formation of nuclear foci by ectopically expressed REV1 was enhanced by the co-expression of Poleta. Importantly, the endogenous REV1 protein accumulated at the UV-irradiated areas of nuclei in Poleta-expressing cells but not in Poleta-deficient XP-V cells. UV-irradiation induced nuclear foci of REV1 and Poleta proteins in both S-phase and G1 cells, suggesting that these proteins may function both during and outside S phase. We reconstituted XP-V cells with wild-type Poleta or with Poleta mutants harboring substitutions in phenylalanine residues critical for interaction with REV1. The REV1-interaction-deficient Poleta mutant failed to promote REV1 accumulation at sites of UV-irradiation, yet (similar to wild-type Poleta) corrected the UV sensitivity of XP-V cells and suppressed UV-induced mutations. Interestingly however, spontaneous mutations of XP-V cells were only partially suppressed by the REV1-interaction deficient mutant of Poleta. Thus, Poleta-REV1 interactions prevent spontaneous mutations, probably by promoting accurate TLS past endogenous DNA lesions, while the interaction is dispensable for accurate Poleta-mediated TLS of UV-induced lesions.

Identification of a novel REV1-interacting motif necessary for DNA polymerase kappa function.

When a replicative DNA polymerase (Pol) is stalled by damaged DNA, a "polymerase switch" recruits specialized translesion synthesis (TLS) DNA polymerase(s) to sites of damage. Mammalian cells have several TLS DNA polymerases, including the four Y-family enzymes (Poleta, Poliota, Polkappa and REV1) that share multiple primary sequence motifs, but show preferential bypass of different DNA lesions. REV1 interacts with Poleta, Poliota, and Polkappa and therefore appears to play a central role during TLS in vivo. Here we have investigated the molecular basis for interactions between REV1 and Polkappa. We have identified novel REV1-interacting regions (RIRs) present in Polkappa, Poliota and Poleta. Within the RIRs, the presence of two consecutive phenylalanines (FF) is essential for REV1-binding. The consensus sequence for REV1-binding is denoted by x-x-x-F-F-y-y-y-y (x, no specific residue and y, no specific residue but not proline). Our results identify structural requirements that are necessary for FF-flanking residues to confer interactions with REV1. A Polkappa mutant lacking REV1-binding activity did not complement the genotoxin-sensitivity of Polk-null mouse embryonic fibroblast cells, thereby demonstrating that the REV1-interaction is essential for Polkappa function in vivo.

Separate roles of structured and unstructured regions of Y-family DNA polymerases.

All organisms have multiple DNA polymerases specialized for translesion DNA synthesis (TLS) on damaged DNA templates. Mammalian TLS DNA polymerases include Pol eta, Pol iota, Pol kappa, and Rev1 (all classified as "Y-family" members) and Pol zeta (a "B-family" member). Y-family DNA polymerases have highly structured catalytic domains; however, some of these proteins adopt different structures when bound to DNA (such as archaeal Dpo4 and human Pol kappa), while others maintain similar structures independently of DNA binding (such as archaeal Dbh and Saccharomyces cerevisiae Pol eta). DNA binding-induced structural conversions of TLS polymerases depend on flexible regions present within the catalytic domains. In contrast, noncatalytic regions of Y-family proteins, which contain multiple domains and motifs for interactions with other proteins, are predicted to be mostly unstructured, except for short regions corresponding to ubiquitin-binding domains. In this review we discuss how the organization of structured and unstructured regions in TLS polymerases is relevant to their regulation and function during lesion bypass.

Initial crystallographic study of human PCNA in complex with a peptide containing the noncanonical PIP-box sequence of human DNA polymerase iota.

Human DNA polymerase iota (Poliota) is one of the Y-family DNA polymerases involved in translesion synthesis (TLS), which allows continued replication at damaged DNA templates. Poliota has a noncanonical PCNA-interacting protein box (PIP-box) within an internal region of the protein. Poliota activity is stimulated by PCNA binding through the noncanonical PIP-box. To clarify the interaction of PCNA with the noncanonical PIP-box of Poliota, PCNA and a Poliota peptide carrying the noncanonical PIP-box complex have been cocrystallized. The crystal belongs to space group C2, with unit-cell parameters a = 167.1, b = 68.7, c = 90.0 A, beta = 95.1 degrees . Structural analysis by molecular replacement is in progress.

Crystallographic study of G178S mutant of human proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is an evolutionarily conserved protein that forms a ring-shaped homotrimer that functions as a sliding clamp for DNA replication. The rev6-1 mutation of Saccharomyces cerevisiae, which inactivates both translesion DNA synthesis and damage-avoidance pathways while having little effect on normal cell growth, has a G178S substitution in the PCNA protein. Human PCNA protein carrying the G178S substitution was crystallized. Two crystal forms were obtained under similar conditions. Crystal forms I and II belong to space groups P2(1), with unit-cell parameters a = 84.1, b = 130.2, c = 97.8 A, beta = 113.4 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 68.1, b = 100.2, c = 131.2 A, respectively. Structural analyses by molecular replacement are now in progress.

DNA damage-induced ubiquitylation of RFC2 subunit of replication factor C complex.

Many proteins involved in DNA replication and repair undergo post-translational modifications such as phosphorylation and ubiquitylation. Proliferating cell nuclear antigen (PCNA; a homotrimeric protein that encircles double-stranded DNA to function as a sliding clamp for DNA polymerases) is monoubiquitylated by the RAD6-RAD18 complex and further polyubiquitylated by the RAD5-MMS2-UBC13 complex in response to various DNA-damaging agents. PCNA mono- and polyubiquitylation activate an error-prone translesion synthesis pathway and an error-free pathway of damage avoidance, respectively. Here we show that replication factor C (RFC; a heteropentameric protein complex that loads PCNA onto DNA) was also ubiquitylated in a RAD18-dependent manner in cells treated with alkylating agents or H(2)O(2). A mutant form of RFC2 with a D228A substitution (corresponding to a yeast Rfc4 mutation that reduces an interaction with replication protein A (RPA), a single-stranded DNA-binding protein) was heavily ubiquitylated in cells even in the absence of DNA damage. Furthermore RFC2 was ubiquitylated by the RAD6-RAD18 complex in vitro, and its modification was inhibited in the presence of RPA. The inhibitory effect of RPA on RFC2 ubiquitylation was relatively specific because RAD6-RAD18-mediated ubiquitylation of PCNA was RPA-insensitive. Our findings suggest that RPA plays a regulatory role in DNA damage responses via repression of RFC2 ubiquitylation in human cells.

Integrating S-phase checkpoint signaling with trans-lesion synthesis of bulky DNA adducts.

Bulky adducts are DNA lesions generated in response to environmental agents including benzo[a]pyrene (a combustion product) and solar ultraviolet radiation. Error-prone replication of adducted DNA can cause mutations, which may result in cancer. To minimize the detrimental effects of bulky adducts and other DNA lesions, S-phase checkpoint mechanisms sense DNA damage and integrate DNA repair with ongoing DNA replication. The essential protein kinase Chk1 mediates the S-phase checkpoint, inhibiting initiation of new DNA synthesis and promoting stabilization and recovery of stalled replication forks. Here we review the mechanisms by which Chk1 is activated in response to bulky adducts and potential mechanisms by which Chk1 signaling inhibits the initiation stage of DNA synthesis. Additionally, we discuss mechanisms by which Chk1 signaling facilitates bypass of bulky lesions by specialized Y-family DNA polymerases, thereby attenuating checkpoint signaling and allowing resumption of normal cell cycle progression.

Rad18 regulates DNA polymerase kappa and is required for recovery from S-phase checkpoint-mediated arrest.

We have investigated mechanisms that recruit the translesion synthesis (TLS) DNA polymerase Polkappa to stalled replication forks. The DNA polymerase processivity factor PCNA is monoubiquitinated and interacts with Polkappa in cells treated with the bulky adduct-forming genotoxin benzo[a]pyrene dihydrodiol epoxide (BPDE). A monoubiquitination-defective mutant form of PCNA fails to interact with Polkappa. Small interfering RNA-mediated downregulation of the E3 ligase Rad18 inhibits BPDE-induced PCNA ubiquitination and association between PCNA and Polkappa. Conversely, overexpressed Rad18 induces PCNA ubiquitination and association between PCNA and Polkappa in a DNA damage-independent manner. Therefore, association of Polkappa with PCNA is regulated by Rad18-mediated PCNA ubiquitination. Cells from Rad18(-/-) transgenic mice show defective recovery from BPDE-induced S-phase checkpoints. In Rad18(-/-) cells, BPDE induces elevated and persistent activation of checkpoint kinases, indicating persistently stalled forks due to defective TLS. Rad18-deficient cells show reduced viability after BPDE challenge compared with wild-type cells (but survival after hydroxyurea or ionizing radiation treatment is unaffected by Rad18 deficiency). Inhibition of RPA/ATR/Chk1-mediated S-phase checkpoint signaling partially inhibited BPDE-induced PCNA ubiquitination and prevented interactions between PCNA and Polkappa. Taken together, our results indicate that ATR/Chk1 signaling is required for Rad18-mediated PCNA monoubiquitination. Recruitment of Polkappa to ubiquitinated PCNA enables lesion bypass and eliminates stalled forks, thereby attenuating the S-phase checkpoint.

Error-prone and inefficient replication across 8-hydroxyguanine (8-oxoguanine) in human and mouse ras gene fragments by DNA polymerase kappa.

Using fragments of human c-Ha-ras and mouse Ha-ras1 genes containing 8-hydroxyguanine (8-OH-G) in hypermutagenic codon 12, we analyzed the kinetics of DNA synthesis catalyzed by human Polkappa. This translesion DNA polymerase, belonging to the Y-family, was found to be moderately inhibited by the presence of 8-OH-G on either mouse or human templates. From our previous results, inhibition of various polymerases by 8-OH-G increases in the following order: Poleta < Polkappa < Polbeta < Polalpha, showing that major replicative and repair polymerases are more sensitive to this lesion than enzymes belonging to the Y-family. In the direct mutagenesis experiments, Polkappa was found to be more mutagenic than Poleta studied previously: it inserted dAMP more efficiently than dCMP opposite 8-OH-G. Polkappa was also able to cause indirect mispair ('action-at-a-distance' mutagenesis), this effect being more distinct on mouse templates. Two adjacent 8-OH-G residues in codon 12 inhibited Polkappa moderately and induced misincorporation of dAMP. However, this effect was not comparable to the strong relaxation of the enzyme specificity, observed previously in the case of Poleta. Polkappa catalyzed incorporation (and misincorporation of dAMP) much more efficiently on mouse templates, human DNA fragments being distinctly worse substrates. Interestingly, in direct mutagenesis systems, the preference for dAMP over dCMP was nearly the same on mouse and human templates.

DNA polymerase kappa is specifically required for recovery from the benzo[a]pyrene-dihydrodiol epoxide (BPDE)-induced S-phase checkpoint.

Previously we identified an intra-S-phase cell cycle checkpoint elicited by the DNA-damaging carcinogen benzo[a]pyrene-dihydrodiol epoxide (BPDE). Here we have investigated the roles of lesion bypass DNA polymerases polkappa and poleta in the BPDE-induced S-phase checkpoint. BPDE treatment induced the re-localization of an ectopically expressed green fluorescent protein-polkappa fusion protein to nuclear foci containing sites of active DNA synthesis in human lung carcinoma H1299 cells. In contrast, a similarly expressed yellow fluorescent protein-poleta fusion protein showed a constitutive nuclear focal distribution at replication forks (in the same cells) that was unchanged in response to BPDE. BPDE-induced formation of green fluorescent protein-polkappa nuclear foci was temporally coincident with checkpoint-mediated S-phase arrest. Unlike "wild-type" cells, Polk(-/-) mouse embryonic fibroblasts (MEFs) failed to recover from BPDE-induced S-phase arrest, while exhibiting normal recovery from S-phase arrest induced by ionizing radiation and hydroxyurea. XPV fibroblasts lacking poleta showed a normal S-phase checkpoint response to BPDE (but failed to recover from the UV light-induced S-phase checkpoint), in sharp contrast to Polk(-/-) MEFs. The persistent S-phase arrest in BPDE-treated Polk(-/-) cells was associated with increased levels of histone gammaH2AX (a marker of DNA double-strand breaks (DSBs)) and activation of the DSB-responsive kinases ATM and Chk2. These data suggest that in the absence of polkappa, replication forks stall at sites of damage and collapse and generate DSBs. Therefore, we conclude that the trans-lesion synthesis enzyme polkappa is specifically required for normal recovery from the BPDE-induced S-phase checkpoint.

Normal immunoglobulin gene somatic hypermutation in Pol kappa-Pol iota double-deficient mice.

Somatic hypermutation (SHM) occurs in the variable region of immunoglobulin genes in germinal center B cells where it plays an important role in affinity maturation of the T cell-dependent immune response. Although the precise mechanism of SHM is still unknown, it has been suggested that error-prone DNA polymerases (Pol) are involved in SHM. Poliota is a member of the error-prone Y-family of DNA polymerases which exhibit translesion synthesis activity in vitro and are highly mutagenic when replicating on non-damaged DNA templates. In BL2 cell line stimulated to induce SHM, the induction is Poliota-dependent. However, in 129-derived strains of mice deficient in Poliota, SHM is normal. One possible explanation for this discrepancy is that a Poliota deficiency in mice might be compensated for by another error-prone DNA polymerase, such as Polkappa, which also belongs to the Y-family of DNA polymerases. Although SHM in Polkappa-deficient mice is normal, their deficiency might be compensated for by Poliota. In this study, we generated Polkappa-Poliota double-deficient mice and examined them for SHM. We found that the double-deficient mice had the normal SHM frequency and profile, rendering them indistinguishable from Polkappa-deficient mice and thus conclude that Poliota and Polkappa are dispensable for SHM in mice.