Mutagenesis Advance Access originally published online on January 4, 2006
Mutagenesis 2006 21(1):49-53; doi:10.1093/mutage/gei073
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Non-transcribed strand repair revealed in quiescent cells
Department of Biology, York University, 4700 Keele Street, Toronto, ON, Canada M3J 1P3
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Abstract |
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Stem cells, one of the progenitors of cancer, exist predominately in a quiescent state. Thus, understanding the mechanisms of DNA repair and mutagenesis in such arrested cells may help unravel the complex process of tumorigenesis. Two major nucleotide excision repair (NER) pathways are known to remove bulky physical or chemical lesions from DNA. Transcription-coupled repair (TCR) acts solely on the transcribed strand of expressed genes, while global genomic repair (GGR) is responsible for the ubiquitous repair of the genome. Indirectly, it has been shown that while TCR functions in quiescent cells GGR does not. To explicitly elucidate this phenomenon, we adapted a quantitative PCR (QPCR) assay to study UV-damage repair via TCR and GGR in quiescent and proliferating cells. We present evidence that repair of untranscribed silent regions of the genome and repair of the non-transcribed strand of active genes proceeds by two discrete mechanisms in quiescent cells; rather than by GGR, which was believed to encompass both. Thus, our findings suggest the existence of an alternate NER pathway in quiescent cells. The proposed subcategories of NER are as follows: (i) TCR, responsible for maintenance of transcribed strands; (ii) GGR, responsible for ubiquitous genome repair; and (iii) non-transcribed strand repair (NTSR), predominantly responsible for the repair of the NTS in arrested cells. In quiescent cells, it is evident that TCR and NTSR function and GGR are arrested. As a consequence, mutation accumulation at temporally silent genes and incomplete or imperfect repair of transcribed genes, in quiescent stem cells, may provide a source of cancer causing mutations.
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Introduction |
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DNA in living cells is continually subjected to chemical alterations. If DNA lesions persist they can lead to mutations, which in turn can contribute to cell death, developmental abnormalities, cancer and other diseases. Multiple DNA repair mechanisms exist to maintain the genetic integrity of cells and can reduce cancer incidence by as much as 10 000-fold (1
); however, these systems have been delineated primarily in actively dividing cells.
The majority of cells in the body, including stem cells, exist in a quiescent state; thus, it is here where most DNA damage is sustained. Given that stem cells are also the only permanent residents in self-renewing tissue populations (2,3), it is logical to assume that most of the multiple cancerous mutations must arise in arrested cells (4–7). Thus, understanding the process of mutagenesis in arrested cells may help to elucidate the complex process of carcinogenesis.
The two major nucleotide excision repair (NER) pathways that remove bulky chemical lesions from DNA are (i) transcription-coupled repair (TCR), which acts solely on the transcribed strand of expressed genes, and (ii) global genomic repair (GGR), which is understood to repair both the non-transcribed strand of expressed genes and areas of the genome that are transcriptionally silent (8). Previous work from our laboratory has demonstrated the lack of DNA repair at an untranscribed transgene in quiescent cells, consistent with a lack of DNA damage recognition at silent regions of the genome (9,10). Evidence, however, suggested that DNA repair proceeds at actively transcribed genes in these same arrested cells (10). Thus, it appeared that GGR is arrested in quiescent cells whereas TCR still functions (10).
To further elucidate this phenomenon, we adapted a repair assay that employed quantitative PCR (QPCR) (11–13) for use with real-time PCR to study UV-damage repair at two endogenous genes, one transcribed and the other not, and at an untranscribed transgene in the same cells. The basis for this assay is that lesions present on the template molecule during PCR block the progression of Taq DNA polymerase (14–16). As a result, blocking lesions, including UV-induced pyrimidine dimers, decrease the percentage of available template molecules for use in PCR and thus decrease the rate of amplification for any given reaction. This rate can be quantified via real-time PCR with a fluorescent reporter (17,18). By recording the amount of fluorescence emission at each cycle, it is possible to monitor the PCR during the exponential phase where the increase in the amount of PCR product correlates with the initial amount of target template. Given that only a single UV-blocking lesion per strand is required to prevent template amplification, the QPCR damage assay enumerates the undamaged template molecules. The restoration of the amplification signal corresponds to DNA repair; and the rate at which this occurs following treatment is a measure of the repair kinetics at the mutational target genes.
Utilizing this real-time QPCR assay, here we report the direct measure of the repair kinetics of low dose UV-induced damage at both transcribed and silent genes in quiescent and proliferating cells. Our results clearly show that in arrested cells, the repair of silent regions of the genome and repair of non-transcribed strands of active genes proceeds by two separate pathways rather than what is currently understood as GGR, thus, demonstrating that an alternate NER pathway must exist in quiescent cells.
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Materials and methods |
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Primary cell strains
C57BL/6 mice (Taconic) were crossed with Big BluBL/6 homozygous mice (Stratagene), transgenic for
80 copies of the 
LIZ insert. F1 embryos were isolated after 12 days post-conception and used to establish primary cell strains, as described in Ref. (19). The resulting embryonic cells were grown for three passages, at which time an aliquot was harvested for PCR-based genotypic sex determination. All experimental trials were performed in three separately derived male cell strains hemizygous for the transgenic target in three independently conducted experiments.
Cell culture
Primary embryonic cells were cultured in DMEM (GIBCO) containing 10% (v/v) fetal bovine serum (FBS; GIBCO) and 1% L-glutamine/penicillin/streptomycin (GIBCO). An atmosphere of 5–7% CO2 was maintained in a humidified incubator at 37°C. Stock cultures were grown in 150 mm tissue culture dishes (Sarstedt, PQ, Canada). To induce quiescence, cultures were seeded in 100 mm dishes (Sarstedt) with 1.2 x 106 cells in the presence of serum and incubated overnight. After a 24 h growth period, the cultures were washed three times with phosphate-buffered saline (PBS) and reincubated with serum-free medium (DMEM, 1% L-glutamine and 1% penicillin/streptomycin). The serum-free medium was renewed at 4 day intervals. After 12 days of culture in serum-free medium, quiescent cells and proliferating cells were irradiated.
Quantitative RT–PCR
RNA was isolated from proliferating and quiescent cell strains following trypsinization by using the absolutely RNATM RT–PCR Miniprep Kit (Stratagene). Briefly, 5 x 106 cells were re-suspended by repeated pipetting in 350 µl lysis buffer supplemented with 2.5 µl ß-mercaptoethanol (14.2 M) and RNA was isolated in accordance with the manufacturer's directions. A small aliquot of RNA was diluted in water and the optical density (at 260 and 280 nm) was determined in addition to gel electrophoresis to quantitate and qualify the RNA. Relative expression of hprt and ß-actin in proliferating and quiescent cells was determined with the aid of the SYBR® Green Quantitative RT–PCR Kit (Sigma) using 100 ng total RNA and 200 nM of the appropriate primers: 5'-CAG GCC AGA CTT TGT TGG AT (hprt RT fwd); 5'-TTGCGCTCATCTTAGGCTTT-3' (hprt RT rev); 5'-TGTTACCAACTGGGACGACGACA-3' (actin RT fwd); and 5'-CTGGGTCATCTTTTCACGGT-3' (actin RT rev), according to the manufacturer's instructions. First strand synthesis was carried out with 1:10 diluted DuraScript RT (Sigma) at 48°C for 30 min followed by denaturation/RT inactivation at 94°C for 2 min and 40 cycles of denaturation (94°C for 15 s) annealing (60°C for 30 s), and extension (72°C for 1 min). Real-time fluorescence detection was performed with the ABI Prism 7700 Sequence Detection System and the absolute (hprt and actin) and relative (hprt) quantification of target RNA was determined from the corresponding ROX normalized CT values from triplicate replicated qRT–PCR.
UV-irradiation
Quiescent and proliferating cells were washed with PBS and irradiated with UV using a germicidal lamp (254 nm, 30 W; General Electric, USA). Cells were either harvested immediately or allowed to recover before DNA isolation, as indicated.
DNA isolation
Cells were pelleted and re-suspended in proteinase K solution (2 mg/ml; Sigma). Genomic DNA was purified from the cell suspension after an incubation time of 2 h at 55°C, followed by phenol/chloroform (1:1) extraction and precipitation with ethanol. The precipitated DNA was spooled on to a hooked glass Pasteur pipette, air dried, and dissolved in 50 µl Tris–HCl, EDTA (TE) buffer (pH 8.0). The DNA was allowed to dissolve overnight at room temperature. The concentration of DNA was then determined with the bisBENZIMIDE (Hoechst 33258) fluorescence assay (Sigma) in accordance with the manufacturer's instructions. After dilution to 20 ng/µl with TE, the DNA concentrations were measured again to ensure accuracy. Samples were then further diluted to 5 ng/µl with TE for use in QPCR.
QPCR conditions
Reaction mixtures (50 µl) contained 20 ng of genomic DNA and 800 nM of the appropriate primers: 5'-CGCCGCCTTGCCCTCGTCT-3' (lambda fwd), 5'-AGCTCCGCAAATTCGCCTACAC-3' (lambda rev); 5'-TGAGAAGGTGGTGGCTGCTGTTGC-3' (ß-globin fwd), 5'-GCACATGCTGCCCATGTGTGTGTG-3' (ß-globin rev); 5'-GGTTCAGGGCCAGAAGCAGACACC-3' (hprt fwd), 5'-GGCAGATGGCCACAGGACTAGAACACC-3' (hprt rev). The QPCR mixtures contained 0.05 U/µl JumpStartTM AccuTaqTM DNA polymerase (Sigma), 50 mM Tris–HCl, 15 mM ammonium sulphate (pH 9.3), adjusted with NH4OH, 2.5 nM MgCl2 and 1% Tween-20, 500 µM dNTP mix (Sigma), and 1 µl of 1:10 000 dilution of SYBR® green I (Sigma) in ddH2O. To amplify the 12.5 kb lambda, 12.5 kb ß-globin, and 12.9 kb hprt fragments, thermal cycling was carried out on the ABI Prism 7700 Sequence Detection System. The samples were amplified as follows: initial denaturation at 96°C for 1 min, 20 cycles of 94°C for 15 s, 72°C for 16 min, followed by an additional 15 cycles of 94°C for 15 s, and 70°C for 16 min plus 20 s extension time per cycle. Samples were held at 70°C for 30 min and stored at 4°C. As verified by standard agarose gel electrophoresis and melting curve analysis, all amplification reactions were extremely precise. Thus, the measured SYBR® green I fluorescence signal was specific to the desired amplicon.
Quantification of PCR amplification
Real-time fluorescence detection was performed with the ABI Prism 7700 Sequence Detection System. Amplification products were quantified from the 
Rn SYBR reporter signal (at the central exponential phase amplification point for each target) following normalization with the passive reference dye (ROX) and after subtraction from the baseline signal generated during the third to the tenth cycle of QPCR. Assuming a random distribution of lesions within the amplicon, the Poisson equation [f(x) = e–x/x!] for undamaged templates is f(0) = e–. The average lesion frequency for each amplicon () is calculated by dividing the normalized amplification signal from UV-treated samples (Rnhv) by the corresponding signal from an equal amount of undamaged genomic DNA (Rn0), = –ln(Rnhv/Rn0). All the experiments were repeated in triplicate in culture and subsequently assayed in triplicate. Given that each treated sample had an analogous control, the established average lesion frequency for each graphical data point was calculated from 18 separate QPCRs.
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Results |
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UV-damaged DNA cannot serve as a template for QPCR amplification because UV-induced lesions effectively block DNA synthesis by Taq polymerase (16
,20). The percentage of template molecules containing blocking lesions within a defined amplicon can be determined using long fragment real-time QPCR, and from this, the number of polymerase blocking lesions quantified (13). Transgenic primary mouse embryonic fibroblasts were irradiated with 0, 2.5, 5, 10 and 20 J/m2 of UV-light. The isolated genomic DNA was used as a template to amplify a 12.5 kb fragment from both the non-transcribed lambda transgene (21,22), non-transcribed endogenous ß-globin gene (23), as well as a 12.9 kb fragment from the transcribed hprt gene. The Rn SYBR reporter signal, during the central exponential phase amplification point, following ROX normalization and baseline subtraction, was used to calculate the relative amounts of undamaged template in each reaction. As expected, an exponential reduction in PCR product (relative fluorescence) with increasing dose was observed during the central exponential phase amplification point for each target (Figure 1A). Assuming a random distribution of lesions, the average number of lesions per kb was determined by dividing the quantity of amplification of UV-treated samples by the amplification of untreated control samples for each fragment (Figure 1B). It should be noted that although all mutational target genes accumulate DNA damage linearly with increasing dose, there are minor UV-sensitivity differences among targets. This disparity is likely the result of genome positional effects and chromosome structure. For example, the highly condense chromatin structure associated with the transgene (24) might explain its relative protection from UV-induced lesions (Figure 1B).
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We used the real-time QPCR assay to measure rates of repair at the transcribed hprt gene, untranscribed ß-globin gene cluster and lambda transgene in proliferating cells. The isolated genomic DNA from UV-treated cells was used as a template to amplify 12.5 kb lambda, 12.5 kb ß-globin and 12.9 kb hprt fragments. By assuming a random distribution of lesions within the amplicon, the Poisson equation can be used to determine the average lesion frequency at each time point. As expected in proliferating cells, the majority of UV-induced lesions were removed from all target genes 24 h following UV-treatment (Figure 2), even though cell doubling times were slowed from 24 to 38 h following treatment. As measured by absolute cloning efficiency (9
), cell viability following irradiation remained high (70 ± 3%).
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When we measured the rate of repair in quiescent cells, a discrepancy with our current understanding of NER was revealed (Figure 3). Consistent with previous reports (9
,10) no repair was observed in quiescent cells at the lambda transgenic target. Moreover, the non-transcribed endogenous ß-globin gene also remained unrepaired in quiescent cells. Hprt in quiescent cells is transcribed (relative mRNA; comparatively to the constitutive expression of ß-actin) at a rate (20.5 ± 3.5) analogous to that of their proliferating counterparts (24.5 ± 6.2). In contrast to the lack of repair of both ß-globin and the lambda transgenic target, UV-damage present on the transcribed hprt gene is efficiently repaired. Cell viability following UV-irradiation was similar, although slightly higher, than that of proliferating cells (79 ± 5%).
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If only those lesions present on the transcribed strand of the hprt gene were repaired by TCR, then the maximal percentage of repair could not exceed 50, which clearly is not the case. Thus, the majority of UV-damage was repaired on both strands of the transcribed hprt gene, but not from either one of the non-transcribed genes during 72 h of quiescence.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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We used a QPCR assay to measure rates of repair at the transcribed hprt gene, untranscribed ß-globin gene cluster and lambda transgene in both proliferating and quiescent cells. A disparity with our current understanding of NER was revealed when we quantified the rates of UV-damage repair in quiescent cells. In proliferating cells, the majority of UV-induced damage appears to be efficiently repaired 24 h post-treatment. However, this is an overestimate of the rate of repair; as even in the absence of UV-lesion removal, DNA synthesis alone can reduce the proportional amount of DNA damage to 50% in one cell doubling. Nonetheless, since the kinetics of repair is similar for all three mutational targets, it appears that TCR is not a dominant factor under these conditions.
In quiescent cells, the data demonstrates an absence of UV-induced lesion repair at untranscribed genes. This finding further establishes the requirement for proliferation for repair at untranscribed regions of the genome (9,10). By definition TCR occurs only in the transcribed strand, whereas the non-transcribed strand is repaired via global genome repair (25). Remarkably, quiescent cells proficiently repaired not only the transcribed strand of the hprt gene but also the non-transcribed strand. Thus, repair of untranscribed regions of the genome and repair of the non-transcribed strand of active genes must proceed by two separate mechanisms, rather than by what is presently understood as the GGR pathway. Moreover, it is postulated that the biphasic kinetics of UV-induced lesion removal (Figure 3) results from differing rates of repair between the transcribed and the non-transcribed stand of the hprt gene.
A similar result has been reported previously in terminally differentiated human neurons (26,27), where GGR appeared to be attenuated in these cells although cyclobutane dimers were removed efficiently from both strands of transcribed genes. The authors tentatively termed this phenomenon differentiation-associated repair (DAR), and suggest exclusivity in terminally differentiated cells (26). Our data indicate that this phenomenon is not restricted to terminally differentiated cells. The data suggest that NER proceeds in at least three separate pathways in quiescent mammalian cells, and propose the following repair nomenclature: (i) TCR, responsible for maintenance of transcribed strands; (ii) GGR, responsible for ubiquitous genome repair, and (iii) non-transcribed strand repair (NTSR), predominantly responsible for the repair of the NTS in arrested cells. It is likely that these three subpathways of NER are functioning in proliferating cells, though their resolution is difficult as replication forks and GGR functionality confound the discrimination of GGR and NTSR pathways.
Differences in repair rates can result from alternate DNA damage recognition mechanisms. The lack of repair at untranscribed genes is thought to be a result of the inaccessibility of DNA in chromatin to damage sensors (9). However, this is contentious as evidence suggested that chromatin structure did not affect GGR abrogation in terminally differentiated human neurons (27). It has been hypothesized previously (10,28), and the data presented here support the fact that GGR is coupled to DNA replication and thus restricted to cells in S phase. Thus, it is when GGR is attenuated during quiescence that it is easy to discriminate NTSR as a separate NER pathway. Moreover, further characterization of the relationships among chromatin composition, DNA synthesis and DNA damage recognition may permit better understanding of lesion stability at various regions throughout the genome, and continued study of repair in non-proliferating cells may allow a more complete understanding of the mechanisms of GGR and NTSR initiation.
In conclusion, GGR is attenuated in quiescent cells, whereas TCR responsible for the repair of the transcribed stand of active genes, and NTSR responsible for the repair of its template proceed in the absence of proliferation.
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Acknowledgments |
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The author is grateful to John Heddle for guidance and support throughout these studies, and greatly appreciate the valuable advice and critical reading of this manuscript by Drs Ann Blank, Philip Hanawalt, Lawrence Loeb and Richard Setlow. Special thanks to Lorien Newell for helpful review and editorial assistance. This work was supported by a grant awarded to John A. Heddle from the National Cancer Institute of Canada (NCIC) with funds from the Canadian Cancer Society. The ABI Prism 7700 Sequence Detection System was purchased with funds from the Canada Foundation for Innovation (CFI) and technical support is provided through a grant from the Canadian Institutes for Health research (CIHR). During completion of this work the author was supported by a fellowship from the National Sciences and Engineering Research Council of Canada (NSERC).
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Notes |
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* To whom correspondence should be addressed at Department of Pathology, University of Washington School of Medicine, HSB K-056, Box 357705, Seattle, WA 98195-7705, USA. Tel: +1 206 543 0556; Fax: +1 206 543 3967; Email: jbielas{at}u.washington.edu
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References |
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Received on September 22, 2005; revised on December 1, 2005; accepted on December 2, 2005.
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