Germline mutation rates at tandem repeat loci in DNA-repair deficient mice

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of non-exposed and irradiated severe combined immunodeficient (scid) and poly(ADP-ribose) polymerase (PARP-1-/-) deficient male mice. Non-exposed scid and PARP-/- male mice showed considerably elevated ESTR mutation rates, far higher than those in wild-type isogenic mice and other inbred strains. The irradiated scid and PARP-1-/- male mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated wild-type isogenic males. ESTR mutation spectra in the scid and PARP-1-/- strains did not differ from those in the isogenic wild-type strains. Considering these data and the results of previous studies, we propose that a delay in repair of DNA damage in scid and PARP-1-/- mice could result in replication fork pausing which, in turn, may affect ESTR mutation rate in the non-irradiated males. The lack of mutation induction in irradiated scid and PARP-1-/- can be explained by the high cell killing effects of irradiation on the germline of deficient mice.     

Expanded simple tandem repeat (ESTR) mutation induction in the male germline: lessons learned from lab mice

Expanded simple tandem repeat (ESTR) DNA loci that are unstable in the germline have provided the most sensitive tool ever developed for investigating low-dose heritable mutation induction in laboratory mice. Ionizing radiation exposures have shown that ESTR mutations occur mainly in pre-meiotic spermatogonia and stem cells. The average spermatogonial doubling dose is 0.62-0.69 Gy for low LET, and 0.18-0.34 Gy for high LET radiation. Chemical alkylating agents also cause significant ESTR mutation induction in pre-meiotic spermatogonia and stem cells, but are much less effective per unit dose than radiation. ESTR mutation induction efficiency is maximal at low doses of radiation or chemical mutagens, and may decrease at higher dose ranges. DNA repair deficient mice (SCID and PARP-1) with elevated levels of single and double-strand DNA breaks have spontaneously elevated ESTR mutation frequencies, and surprisingly do not show additional ESTR mutation induction following irradiation. In contrast, ESTR mutation induction in p53 knock-outs is indistinguishable from that of wild-type mice. Studies of sentinel mice exposed in situ to ambient air pollution showed elevated ESTR mutation frequencies in males exposed to high levels of particulate matter. These studies highlight the application of the ESTR assay for assessing environmental hazards under real-world conditions. All ESTR studies to date have shown untargeted mutations that occur at much higher frequencies than predicted. The mechanism of this untargeted mutation induction is unknown, and must be elucidated before we can fully understand the biological significance of ESTR mutations, or use these markers for formal risk assessment. Future studies should focus on the mechanism of ESTR mutation induction, refining dose responses, and developing ESTR markers for other animal species.     

No correlation between germline mutation at repeat DNA and meiotic crossover in male mice exposed to X-rays or cisplatin

To test the hypothesis that mouse germline expanded simple tandem repeat (ESTR) mutations are associated with recombination events during spermatogenesis, crossover frequencies were compared with germline mutation rates at ESTR loci in male mice acutely exposed to 1 Gy of X-rays or to 10 mg/kg of the anticancer drug cisplatin. Ionising radiation resulted in a highly significant 2.7–3.6-fold increase in ESTR mutation rate in males mated 4, 5 and 6 weeks after exposure, but not 3 weeks after exposure. In contrast, irradiation had no effect on meiotic crossover frequencies assayed on six chromosomes using 25 polymorphic microsatellite loci spaced at approximately 20 cM intervals and covering 421 cM of the mouse genome. Paternal exposure to cisplatin did not affect either ESTR mutation rates or crossover frequencies, despite a report that cisplatin can increase crossover frequency in mice.

Correlation analysis did not reveal any associations between the paternal ESTR mutation rate and crossover frequency in unexposed males and in those exposed to X-rays or cisplatin. This study does not, therefore, support the hypothesis that mutation induction at mouse ESTR loci results from a general genome-wide increase in meiotic recombination rate.     

The effects of MSH2 deficiency on spontaneous and radiation-induced mutation rates in the mouse germline

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of mismatch repair deficient Msh2 knock-out mice. Spontaneous mutation rates in homozygous Msh2(-/-) males were significantly higher than those in isogenic wild-type (Msh2(+/+)) and heterozygous (Msh2(+/-)) mice. In contrast, the irradiated Msh2(-/-) mice did not show any detectable increases in their mutation rate, whereas significant ESTR mutation induction was observed in the irradiated Msh2(+/+) and Msh2(+/-) animals. Considering these data and the results of other publications, we propose that the Msh2-deficient mice possess a mutator phenotype in their germline and somatic tissues while the loss of a single Msh2 allele does not affect the stability of heterozygotes.     

The effects of methyl-donor deficiency on mutation induction and transgenerational instability in mice

The results of recent human and animal studies have provided strong evidence for the epigenetic effects of a dietary deficiency of methyl donors such as folate, choline and methionine on cancer risk and some other common diseases. However, the mechanisms underlying the links between epigenetic alterations and disease remain elusive. To establish whether a methyl-donor deficient diet can result in long-term changes in mutation rate in treated animals and their offspring, BALB/c male mice were maintained for 8 weeks, from 4 weeks of age, on a synthetic diet lacking in choline and folic acid. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in sperm samples of treated males, as well as in sperm and brain of their first-generation offspring. ESTR mutation frequency in the germline of males sacrificed immediately after treatment or sampled 6 and 10 weeks after the end of dietary restriction did not significantly differ from that in age-matched control groups. The frequency of ESTR mutation in DNA samples extracted from sperm and brain of the first-generation offspring of treated mice was also similar to that in controls. The results of our study suggest that the effects of a methyl-donor deficient diet on mutation induction and transgenerational instability in mice are likely to be negligible.     

Radiation-induced mutation at tandem repeat DNA Loci in the mouse germline: spectra and doubling doses

The spectra and dose response for mutations at expanded simple tandem repeat (ESTR) loci in the germline of male mice acutely exposed to low-LET X or gamma rays at pre-meiotic stages of spermatogenesis were compared in five strains of laboratory mice. Most mutation events involved the gain or loss of a relatively small number of repeat units, and the distributions of length changes were indistinguishable between the exposed and control males. Overall, a significant bias toward gains of repeats was detected, with approximately 60% of mutants showing gains. The values for ESTR mutation induction did not differ substantially between strains. The highest values of doubling dose were obtained for two genetically related strains, BALB/c and C.B17 (mean value 0.98 Gy). The estimates of doubling dose for three other strains (CBA/H, C57BL/6 x CBA/H F1 and 129SVJ x C57BL/6) were lower, with a mean value of 0.44 Gy. The dose response for ESTR mutation across all five strains was very close to that for the specific loci (Russell 7-locus test). The mechanisms of ESTR mutation induction and applications of this system for monitoring radiation-induced mutation in the mouse germline are discussed.     

The effects of maternal irradiation during adulthood on mutation induction and transgenerational instability in mice

The long-term genetic effects of maternal irradiation remain poorly understood. To establish the effects of radiation exposure on mutation induction in the germline of directly exposed females and the possibility of transgenerational effects in their non-exposed offspring, adult female BALB/c and CBA/Ca mice were given 1Gy of acute X-rays and mated with control males. The frequency of mutation at expanded simple tandem repeat (ESTR) loci in the germline of directly exposed females did not differ from that of controls. Using a single-molecule PCR approach, ESTR mutation frequency was also established for both germline and somatic tissues in the first-generation offspring of irradiated parents. While the frequency of ESTR mutation in the offspring of irradiated males was significantly elevated, maternal irradiation did not affect stability in their F(1) offspring. Considering these data and the results of our previous study, we propose that, in sharp contrast to paternal exposure to ionising radiation, the transgenerational effects of maternal high-dose acute irradiation are likely to be negligible.     

Germline mutation induction at mouse repeat DNA loci by chemical mutagens

Mutation rates at two expanded simple tandem repeat (ESTR) loci were studied in the germline of male mice exposed to two monofunctional alkylating agents, ethylnitrosourea (ENU) and isopropyl methanesulfonate (iPMS), and a topoisomerase II inhibitor, etoposide. Pre-meiotic exposure to the alkylating agents resulted in a highly significant increase in ESTR mutation rate, but did not alter post-meiotically exposed cells. Pre-meiotic mutation induction by ENU and iPMS was linear within the interval of doses from 12.5 to 25mg/kg and reached a plateau at higher concentrations. Paternal exposure to etoposide resulted in ESTR mutation induction at meiotic stages but did not affect post- or pre-meiotic cells. The pattern of ESTR mutation induction after pre-meiotic and meiotic exposure to chemical mutagens was similar to that previously obtained by various traditional approaches for monitoring germline mutation in mice. The results of this study show that ESTR loci provide a new efficient experimental system for monitoring the genetic effects of chemical mutagens, capable of detecting increases in mutation rates at low doses of exposure.     

Stage-specificity of spontaneous mutation at a tandem repeat DNA locus in the mouse germline

Mouse expanded simple tandem repeat (ESTR) loci are the most unstable loci in the mouse genome. Despite the fact that over the last decade these loci have been extensively used for studying germline mutation induction in mice, to date little is known about the mechanisms underlying spontaneous and induced ESTR mutation. Here we used flow cytometry and single-molecule PCR to compare the frequency of ESTR mutation in four flow-sorted fractions of the mouse male germ cells – spermatogonia, spermatocytes I, round and elongated spermatids. The frequency and the spectrum of ESTR mutation did not significantly differ between different stages of mouse spermatogenesis. Considering these data and the results of other publications, we propose that spontaneous ESTR mutation is mostly attributed to replication slippage in spermatogonia and these loci may be regarded as a class of expanded microsatellites.     

A single-molecule PCR approach to the measurement of induced expanded simple tandem repeat instability in vitro

Sensitive and precise models are needed to identify potential genotoxicity at environmentally relevant doses of mutagens. The size length alterations in expanded simple tandem repeat (ESTR) loci have been used as a biomarker of genetic instability caused by a variety of agents in the mouse germline. The mechanisms operating in both spontaneous and induced instability are poorly understood. We have developed a single-molecule polymerase chain reaction (SM-PCR) method to investigate mutation at the mouse ESTR locus Ms6-hm in the murine C3H/10T1/2 embryonic cell line. Growth of cells to levels of high cell density induced increased ESTR instability, with mutation frequencies 5.1-fold (+/-2.8) over sub-confluent cultures. Accordingly, cell cultures were maintained at sub-confluent levels for further investigations of the induction of ESTR mutation by genotoxic agents. Treatment with the DNA alkylating agent N-nitroso-N-ethylurea (ENU) resulted in a 1.94-fold (+/-1.1) increase in mutation frequency, similar to responses measured previously in the germline in vivo. Therefore, mutagen exposure can also affect somatic (non-meiotic) rapidly dividing mouse cells. This SM-PCR approach eliminates the requirement of sub-cloning individual treated cells, thereby, reducing the time needed to screen for ESTR mutation, and will be a very useful tool for future investigations into the mechanisms involved in ESTR mutation.     

Germline mutation rates in mice following in utero exposure to diesel exhaust particles by maternal inhalation

The induction of inherited DNA sequence mutations arising in the germline (i.e., sperm or egg) of mice exposed in utero to diesel exhaust particles (DEPs) via maternal inhalation compared to unexposed controls was investigated in this study. Previous work has shown that particulate air pollutants (PAPs) from industrial environments cause DNA damage and mutations in the sperm of adult male mice. Effects on the female and male germline during critical stages of development (in utero) are unknown. In mice, previous studies have shown that expanded simple tandem repeat (ESTR) loci exhibit high rates of spontaneous mutation, making this endpoint a valuable tool for studying inherited mutation and genomic instability. In the present study, pregnant C57Bl/6 mice were exposed to 19mg/m(3) DEP from gestational day 7 through 19, alongside air exposed controls. Male and female F1 offspring were raised to maturity and mated with control CBA mice. The F2 descendents were collected and ESTR germline mutation rates were derived from full pedigrees (mother, father, offspring) of F1 male and female mice. We found no evidence for increased ESTR mutation rates in females exposed in utero to DEP relative to control females. In contrast, a statistically significant increase in the mutation frequency of male mice exposed in utero to DEP was observed (2-fold; Fisher's exact p<0.05). Thus, maternal exposure to DEP results in increased mutation in sperm during development.     

The dose and dose-rate effects of paternal irradiation on transgenerational instability in mice: a radiotherapy connection

The non-targeted effects of human exposure to ionising radiation, including transgenerational instability manifesting in the children of irradiated parents, remains poorly understood. Employing a mouse model, we have analysed whether low-dose acute or low-dose-rate chronic paternal γ-irradiation can destabilise the genomes of their first-generation offspring. Using single-molecule PCR, the frequency of mutation at the mouse expanded simple tandem repeat (ESTR) locus Ms6-hm was established in DNA samples extracted from sperm of directly exposed BALB/c male mice, as well as from sperm and the brain of their first-generation offspring. For acute γ-irradiation from 10-100 cGy a linear dose-response for ESTR mutation induction was found in the germ line of directly exposed mice, with a doubling dose of 57 cGy. The mutagenicity of acute exposure to 100 cGy was more pronounced than that for chronic low-dose-rate irradiation. The analysis of transgenerational effects of paternal irradiation revealed that ESTR mutation frequencies were equally elevated in the germ line (sperm) and brain of the offspring of fathers exposed to 50 and 100 cGy of acute γ-rays. In contrast, neither paternal acute irradiation at lower doses (10-25 cGy), nor low-dose-rate exposure to 100 cGy affected stability of their offspring. Our data imply that the manifestation of transgenerational instability is triggered by a threshold dose of acute paternal irradiation. The results of our study also suggest that most doses of human exposure to ionising radiation, including radiotherapy regimens, may be unlikely to result in transgenerational instability in the offspring children of irradiated fathers.     

Detection of induced male germline mutation: correlations and comparisons between traditional germline mutation assays, transgenic rodent assays and expanded simple tandem repeat instability assays

Several rodent assays are capable of monitoring germline mutation. These include traditional assays, such as the dominant lethal (DL) assay, the morphological specific locus (SL) test and the heritable translocation (HT) assay, and two assays that have been developed more recently--the expanded simple tandem repeat (ESTR) and transgenic rodent (TGR) mutation assays. In this paper, we have compiled the limited amount of experimental data that are currently available to make conclusions regarding the comparative ability of the more recently developed assays to detect germline mutations induced by chemical and radiological agents. The data suggest that ESTR and TGR assays are generally comparable with SL in detecting germline mutagenicity induced by alkylating agents and radiation, though TGR offered less sensitivity than ESTR in some cases. The DL and HT assays detect clastogenic events and are most susceptible to mutations arising in post-spermatogonial cells, and they may not provide the best comparisons with TGR and ESTR instability. The measurement of induced ESTR instability represents a relatively sensitive method of identifying agents causing germline mutation in rodents, and may also be useful for bio-monitoring exposed individuals in the human population. Any future use of the TGR and ESTR germline mutation assays in a regulatory testing context will entail more robust and extensive characterization of assay performance. This will require substantially more data, including experiments measuring multiple endpoints, a greatly expanded database of chemical agents and a focus on characterizing stage-specific activity of mutagens in these assays, preferably by sampling epididymal sperm exposed at defined pre-meiotic, meiotic and post-meiotic stages of development.     

Inhibition of human cytochrome P450 1B1, 1A1 and 1A2 by antigenotoxic compounds,purpurin and alizarin

Expanded simple tandem repeat (ESTR) loci include some of the most unstable DNA in the mouse genome and have been extensively used in pedigree studies of germline mutation. We now show that repeat DNA instability at the mouse ESTR locus Ms6-hm can also be monitored by single molecule PCR analysis of genomic DNA. Unlike unstable human minisatellites which mutate almost exclusively in the germline by a meiotic recombination-based process, mouse Ms6-hm shows repeat instability both in germinal (sperm) DNA and in somatic (spleen, brain) DNA. There is no significant variation in mutation frequency between mice of the same inbred strain. However, significant variation occurs between tissues, with mice showing the highest mutation frequency in sperm. The size spectra of somatic and sperm mutants are indistinguishable and heavily biased towards gains and losses of only a few repeat units, suggesting repeat turnover by a mitotic replication slippage process operating both in the soma and in the germline. Analysis of male mice following acute pre-meiotic exposure to X-rays showed a significant increase in sperm but not somatic mutation frequency, though no change in the size spectrum of mutants. The level of radiation-induced mutation at Ms6-hm was indistinguishable from that established by conventional pedigree analysis following paternal irradiation. This confirms that mouse ESTR loci are very sensitive to ionizing radiation and establishes that induced germline mutation results from radiation-induced mutant alleles being present in sperm, rather than from unrepaired sperm DNA lesions that subsequently lead to the appearance of mutants in the early embryo. This single molecule monitoring system has the potential to substantially reduce the number of mice needed for germline mutation monitoring, and can be used to study not only germline mutation but also somatic mutation in vivo and in cell culture.     

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.     

Instability of expanded simple tandem repeats is induced in cell culture by a variety of agents: N-Nitroso-N-ethylurea, benzo(a)pyrene, etoposide and okadaic acid

Expanded simple tandem repeat (ESTR) sequences have proven useful biomarkers to detect genotoxicity in vivo. Their high sensitivity has been used to assess environmentally relevant doses of mutagens such as ionizing radiation, DNA alkylating agents and airborne particulate pollution, for germline mutations in mouse assays. The mutagenic response involves size alteration of these ESTR loci induced by agents causing a variety of cellular damage. The mechanistic aspects of this induced instability remain unclear and have not been studied in detail. Mechanistic knowledge is important to help understand the relevance of increased ESTR mutation frequencies. In this study, we applied a murine cell culture system to examine induced response to four agents exhibiting different modes of toxic action including: N-nitroso-N-ethylurea (ENU), benzo(a)pyrene (BaP), okadaic acid and etoposide at slightly sub-toxic levels. We used single-molecule-polymerase chain reaction (SM-PCR) to assess the relative mutant frequency after 4-week chemical treatments at the Ms6-hm ESTR sequence of cultured C3H/10T1/2 cells (a mouse embryonic cell line). Increased mutation was observed with both 0.64 mM ENU (1.95-fold increase, P<0.0001), 1 microM benzo(a)pyrene (1.87-fold increase, P=0.0006) and 3 nM etoposide (1.89-fold increase, P=0.0003). The putative ESTR mutagen okadaic acid (1.27-fold increase, P=0.2289), administered at 0.5 nM, did not affect the C3H/10T1/2 Ms6-hm locus. Therefore, agents inducing small and bulky adducts, and indirectly causing strand breaks through inhibition of topoisomerase, caused similar induction of instability at an ESTR locus at matched toxicities. As size spectra for induced mutations were identical, the data indicate that although these chemicals exhibit distinct modes of action, a similar indirect process is influencing ESTR instability. In contrast, a potent tumour promoter that is a kinase inhibitor does not contribute to induced ESTR instability in cell culture.     

Activities of human DNA polymerase kappa in response to the major benzo[a]pyrene DNA adduct: error-free lesion bypass and extension synthesis from opposite the lesion

In cells, the major benzo[a]pyrene DNA adduct is the highly mutagenic (+)-trans-anti-BPDE-N(2)-dG. In eukaryotes, little is known about lesion bypass of this DNA adduct during replication. Here, we show that purified human Polkappa can effectively bypass a template (+)-trans-anti-BPDE-N(2)-dG adduct in an error-free manner. Kinetic parameters indicate that Polkappa bypass of the (-)-trans-anti-BPDE-N(2)-dG adduct was approximately 41-fold more efficient compared to the (+)-trans-anti-BPDE-N(2)-dG adduct. Furthermore, we have found another activity of human Polkappa in response to the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts: extension synthesis from mispaired primer 3' ends opposite the lesion. In contrast, the two adducts strongly blocked DNA synthesis by the purified human Polbeta and the purified catalytic subunits of yeast Polalpha, Poldelta, and Pol epsilon right before the lesion. Extension by human Polkappa from the primer 3' G opposite the (+)- and (-)-trans-anti-BPDE-N(2)-dG adducts was mediated by a -1 deletion mechanism, probably resulting from re-aligning the primer G to pair with the next template C by Polkappa prior to DNA synthesis. Thus, sequence contexts 5' to the lesion strongly affect the fidelity and mechanism of the Polkappa-catalyzed extension synthesis. These results support a dual-function model of human Polkappa in bypass of BPDE DNA adducts: it may function both as an error-free bypass polymerase alone and an extension synthesis polymerase in combination with another polymerase.     

Late onset of spermatogenesis and gain of fertility in POG-deficient mice indicate that POG is not necessary for the proliferation of spermatogonia

The germ cell-deficient (gcd) mouse mutation is a recessive, transgenic insertional mutation associated with the disruption of two Chr11 genes, Pog (proliferation of germ cells) and Vrk2 (vaccinia virus-related protein kinase 2). We have recently shown that like gcd/gcd mice, targeted Pog-/- males and females show virtually no spermatogenesis or oogenesis at 4-6 wk of age. Because Pog is deleted in gcd/gcd and Pog-/- mice, a comparison of the phenotypes of the two mouse models is appropriate. Here, we report that unlike in POG-deficient females, the germ cells in POG-deficient males eventually populate the seminiferous tubules at 9 wk, and fertility can be achieved by 12 wk. Homozygous gcd/gcd males did not show a similar degree of germ cell population, and most gcd/gcd males remained infertile at 16 and 22 wk of age. A comparison of the degree of germ cell deficiency at 13.5 days postcoitum and 1 day postpartum between Pog-/- and gcd/gcd males revealed that gcd/gcd males had far fewer germ cells than Pog-/- males at both time points. Our data suggest that Pog is essential for proper primordial germ cell proliferation in the embryonic stage but is not needed for spermatogonial proliferation after birth. Thus, the difference in the spermatogenetic potential in adult Pog-/- and gcd/gcd mice may result from the severity of germ cell deficiency rather than from the inability of gcd/gcd spermatogonia to proliferate efficiently. The greater deficiency of germ cells before the onset of spermatogenesis seen in gcd/gcd males compared to Pog-/- mice suggests either that the different background affects the outcome of Pog deletion or that Vrk2 has additional effects on germ cell development.     

Requirements for the interaction of mouse Polkappa with ubiquitin and its biological significance

Polkappa protein is a eukaryotic member of the DinB/Polkappa branch of the Y-family DNA polymerases, which are involved in the tolerance of DNA damage by replicative bypass. Despite universal conservation through evolution, the precise role(s) of Polkappa in this process has remained unknown. Here we report that mouse Polkappa can physically interact with ubiquitin by yeast two-hybrid screening, glutathione S-transferase pulldown, and immunoprecipitation methods. The association of Polkappa with ubiquitin requires the ubiquitin-binding motifs located at the C terminus of Polkappa. In addition, Polkappa binds with monoubiquitinated proliferating cell nuclear antigen (PCNA) more robustly than with non-ubiquitinated PCNA. The ubiquitin-binding motifs mediate the enhanced association between monoubiquitinated PCNA and Polkappa. The ubiquitin-binding motifs are also required for Polkappa to form nuclear foci after UV radiation. However, the ubiquitin-binding motifs do not affect Polkappa half-life. Finally, we have examined levels of Polkappa expression following the exposure of mouse cells to benzo[a]pyrene-dihydrodiol epoxide or UVB radiation.     

Involvement of vertebrate polkappa in Rad18-independent postreplication repair of UV damage

DNA damage, which is left unrepaired by excision repair pathways, often blocks replication, leading to lesions such as breaks and gaps on the sister chromatids. These lesions may be processed by either homologous recombination (HR) repair or translesion DNA synthesis (TLS). Vertebrate Polkappa belongs to the DNA polymerase Y family, as do most TLS polymerases. However, the role for Polkappa in vertebrate cells is unclear because of the lack of reverse genetic studies. Here, we generated cells deficient in Polkappa (polkappa cells) from the chicken B lymphocyte line DT40. Although purified Polkappa is unable to bypass ultraviolet (UV) damage, polkappa cells exhibited increased UV sensitivity, and the phenotype was suppressed by expression of human and chicken Polkappa, suggesting that Polkappa is involved in TLS of UV photoproduct. Defects in both Polkappa and Rad18, which regulates TLS in yeast, in DT40 showed an additive effect on UV sensitivity. Interestingly, the level of sister chromatid exchange, which reflects HR-mediated repair, was elevated in normally cycling polkappa cells. This implies functional redundancy between HR and Polkappa in maintaining chromosomal DNA. In conclusion, vertebrate Polkappa is involved in Rad18-independent TLS of UV damage and plays a role in maintaining genomic stability.