BACKGROUND
cellular genomic dna constantly suffers from damage induced by various external genotoxic agents and endogenous metabolic materials.
in eukaryotic cells there are multiple conserved dna repair pathways including nucleotide excision repair , which is a dna repair mechanism removing a variety of helix-distorting dna lesions including ultraviolet radiation -induced cyclobutane pyrimidine dimers , 6- <dig> pyrimidine pyrimidone photoproducts , and cigarette smoke-induced benzopyrene dna adducts.
the relevance of this repair pathway is indicated by the observation that defected ner genes can result in rare human autosomal recessive disorders such as xeroderma pigmentosum  and cockayne syndrome   <cit> .
there are two ner sub-pathways: global genomic ner repair  and transcription-coupled repair , which differ mainly in the step of recognition of the dna lesions  <cit> .
tcr preferentially repairs the transcribed strand or transcribed genes compared to the untranscribed strand or silenced genes.
in other words, the transcribed strand or genes that are undergoing transcription exhibit a faster rate of repairing dna damage than the untranscribed strand and the overall genome  <cit> .
rna polymerase ii plays a critical role in the recognition of dna damage in the tcr pathway.
the current tcr model proposes that rna polymerase, stalled at a lesion point, directs the recruitment of repair enzymes to the transcribed strand of an active gene  <cit> .
this model assumes that rna polymerase ii must be removed from the lesion site of the transcribed strand to provide access for the repair complex, which initiates the repair process through unwinding the double helix at the damaged site, removal of the dna terminus, and finally filling the gap and joining the dna strands.
previous studies have shown that tcr is a critical survival pathway protecting against acute toxic and long-term effects of genotoxic exposures  <cit> .
a number of human genetic syndromes such as xeroderma pigmentosum complementing group d and cockayne syndromes a and b  have been identified as associated with a deficient tcr mechanism against uv radiation, and therefore the cells from these patients are sensitive to uv irradiation  <cit> .
in recent years, extensive knowledge regarding tcr has been acquired.
however, as mentioned by the tcr research pioneers, hanawalt and spivak, the more we have learned, the more questions have been raised about the intricate details of tcr and its relevance to human disease  <cit> .
dna dependent protein kinase catalytic subunit  is a member of the phosphatidylinositol 3-kinase-related protein kinase  family with serine/threonine kinase activity, and it is well known as a critical component of the non-homologous end joining  pathway of dna double-strand break  repair  <cit> .
when dsb occurs in the cells, the ku80/ku <dig> heterodimer firstly recognizes and binds to the broken dna, followed by recruitment of dna-pkcs to the damage site to form the dna-pk complex with ku80/ku <dig>  which consequently triggers the damage signal and subsequent dna repair  <cit> .
it has previously been reported that cells with mutated or decreased expression of dna-pkcs are sensitive to uv irradiation  <cit> .
furthermore, mouse scid cells or hamster v- <dig> cells with mutated dna-pkcs exhibit a decreased capability of ner activity, with reduced unscheduled dna synthesis  after uv irradiation  <cit> .
however, this ner deficiency cannot be complemented by addition of dna-pk, suggesting a non-direct role of dna-pk in the ner repair steps.
since rna polymerase ii is a substrate of dna-pkcs  <cit> , it was suggested that dna-pk could act as a dna damage-sensor that might regulate dna repair via transcriptional modulation  <cit> .
in this study, we postulated that dna-pkcs might play a role in the transcription-coupled repair pathway of uv-induced cpds damage.
in order to detect the tcr of ultraviolet  radiation-induced dna damage, we established a novel method of tcr assay based on dna strand-specific pcr  of the dihydrofolate reductase  gene.
we verified the efficiency and feasibility of this tcr assay in a sirna-mediated csb-depleted cell model, in which csb depletion resulted in a deficient tcr pathway.
in addition, tcr of the uv damaged c-myc gene was also found to be attenuated when the c-myc gene was silenced by the treatment of dmso in hl <dig> cells.
using this verified method, we demonstrated that sirna-mediated silencing of dna-pkcs has no effect on the ggner efficiency, while repair efficiency of that transcribed strand  of dhfr gene was significantly lower in dna-pkcs-depleted hela-h <dig> cells than that in control hela-nc cells.
furthermore, we have carried out the co-immunoprecipitation assay and identified an interaction between dna-pkcs and the cyclin t <dig> protein.
cyclint <dig>  together with cyclin-dependent kinase  <dig> , comprises the positive transcription elongation factor b .
the activated cdk <dig> phosphorylates the carboxyl terminal domain  of rna pol ii, and then promotes transcription elongation  <cit> .
hence, we postulate that dna-pkcs may modulate the activity of rna pol ii through associating with cyclin t <dig>  the present study provides new evidence suggesting the involvement of dna-pkcs in the transcription-coupled repair pathway of nucleotide excision repair in response to uv-induced dna damage.
RESULTS
depletion of dna-pkcs enhanced the cellular uv sensitivity
as shown in the growth curve , depletion of dna-pkcs mediated by transfecting with the shrna vector expressing the specific sirna molecules targeting the catalytic motif of the dna-pkcs gene  significantly decreased the growth ability of hela-h <dig> as compared to the control hela-nc cells after exposure to  <dig> j/m <dig>  and  <dig> j/m <dig>  uv irradiation.
to confirm these results, a clonogenic assay was performed, and the cell survival levels also showed an increased sensitivity of the dna-pkcs-depleted hela-h <dig> cells to uv radiation as compared with the control hela-nc cells .
depletion of dna-pkcs does not affect the ggner of uv-induced dna damage
dna damage from uv exposure results in the frequent formation of cyclobutane pyrimidine dimers  and 6- <dig> pyrimidine pyrimidone photoproducts .
the thymine dimer in dna sequences can be effectively recognized and incised by t <dig> endo v. with increased repair time, the residual thymine dimers decrease, and the extent of fragmentation of genomic dna by t <dig> endo v is also decreased.
therefore, detection of genomic fragmentation by the alkaline electrophoresis assay can indicate the whole genomic dna repair efficiency.
as shown in figure  <dig>  the global genome nucleotide excision repair  was completed  <dig> h after uv irradiation, and there was no difference in ggner efficiency between dna-pkcs-depleted cells  and control cells  after  <dig> j/m <dig> uv exposure .
we also compared the ggner efficiency in hela cells possessing a decreased expression of the csb gene.
as shown in figure 2b, depression of the csb expression mediated by sirna also had no obvious effect on ggner in helasirna- <dig> cells as compared to the control hela-nc cells.
the hl <dig> cell line is sensitive to uv radiation, and the alkaline electrophoresis result confirmed that the ggner efficiency of uv-induced dna damage in hl <dig> cells is slower than in hela cells .
although the dna damage was almost completely repaired at  <dig> h after  <dig> j/m <dig> uv radiation in hela cells , that was not accomplished in hl <dig> cells until  <dig> h after exposure .
development and validation of the novel method of tcr assay based on dna strand-specific pcr
although depletion of dna-pkcs sensitizes hela cells to uv radiation, our results show no obvious effect on the efficiency of the ggner activity.
next, we decided to focus on the transcription-coupled repair  of uv-induced dna damage.
the available classical method of tcr assay is time-consuming, requires running sequencing gels, and uses radioactive isotopic probes in southern blotting hybridization <cit> .
here we designed a novel tcr assay  based on strand-specific polymerase chain reaction  with a set of smartly designed primers .
this method partially evolved from the idea of the quantitative pcr detection of transcription-coupled repair based on the chromatin co-immunoprecipitated dna fragments  <cit> , and the primers designing strategy of the suppression subtractive hybridization pcr method  <cit> .
the general theoretical basis and the technical principles are as follows: 1) it is based on the dna strand-specific pcr method to amplify the transcribed strand or untranscribed strand in the separate reaction tube with specifically designed primers for each; 2) the repair efficiency of the transcribed and untranscribed strands is reflected by the pcr efficiency of the synthesized first-strand of the transcribed or untranscribed strand using t <dig> endo v-digested genomic dna as a template, which is directly related to the repair efficiency of cpds located in dna strands.
<dig> the underline sequence is an adaptor universal primer  without homology to mammalian genomic dna.
the experimental steps of this method are outlined in figure 3a: 1) digest the genomic dna extracted from uv exposed cells with t <dig> endo v to nick the dna at the residual cpds damage sites; 2) after dna denaturation, the first-strand dna is synthesized using the specific first-strand synthesis primer , which is composed of an upstream adaptor universal primer  and a downstream annealing primer specifically targeting the active transcribed-strand  or the untranscribed-strand ; 3) dna elution is done to remove the free first-strand primer, and pcr amplification is carried out using the primer pairs of the up  and the strand-specific down-stream primer targeting the first-strand template of the transcribed strand  or the untranscribed strand ; 4) analysis of the pcr products by electrophoresis.
the lower efficiency of repairing the damaged dna strand, the more cpds damage sites remain.
consequently, more residual damage sites provide more nicking sites for t <dig> endo v, which eventually lead to a less amount of intact dna templates for the first-strand synthesis and the pcr amplification.
if both strands of the double-strand dna molecule is intact , the transcribed-strand is amplified logarithmically in the transcribed-strand pcr reaction tube due to the smart pcr primer pair of tsp <dig> and up which is appended into the templates during the first-strand synthesis of the transcribed-strand.
although tsp <dig> can also anneal with the complementary untranscribed-strand, it is amplified linearly due to only one primer available for this untranscribed-strand during pcr reaction .
in the same way, the untranscribed-strand is amplified logarithmically and the transcribed-strand is amplified linearly in the untranscribed-strand pcr reaction tube due to the smart pcr primer pair of ntsp <dig> and up.
therefore, the transcribed strand or untranscribed strand can be easily determined in this method.
concurrently, a β-actin gene fragment without a tt dimer sequence was used as an internal template reference.
in order to validate the reliability and efficiency of this new method, we firstly detected the tcr efficiency of the c-myc gene in uv-irradiated hl- <dig> cells under its different transcriptional status.
both mrna and protein levels of c-myc are overexpressed in hl <dig> cells.
when hl <dig> cells are induced to differentiate by dmso, the transcriptional activity of c-myc is rapidly silenced <cit> .
as shown in figure 4a, c-myc expression in hl <dig> cells is remarkably silenced after the treatment of dmso at  <dig> ~1%  and c-myc expression is almost completely inhibited after 1%  dmso treatment for 48- <dig> h. for the following experiments, hl <dig> cells were treated with 1% dmso for  <dig> h, and then irradiated with  <dig> j/m <dig> uv.
the tcr efficiency of c-myc was then detected using this new method.
the results showed that the repair efficiency of the uv-damaged c-myc transcribed strand in dmso treated cells was significantly decreased as compared with the control cells without dmso treatment .
this result indicates that the new tcr assay method based on strand-specific pcr is reliable for the detection of tcr of dna damage.
we then used this new method to further assess the tcr of uv-induced dna damage in a cockayne syndrome b gene -depleted cell line , which was generated from hela cells by transfecting specific sirna construct to deplete the csb mrna  and protein expression .
csb previously has been shown to play an important role in the tcr pathway.
after  <dig> j/m <dig> uv radiation, strand-specific pcr of endo v enzyme digested transcribed and untranscribed strands of dhfr gene was performed.
as shown in figure 5c, no difference in pcr efficiency of the untranscribed strand was observed between helasirna- <dig> and the control cells, but the pcr efficiency of the transcribed strand in helasirna- <dig> cells was obviously lower compared to that in the control cells at all repair time points post uv-irradiation.
it seems that the repair was not accomplished even at  <dig> h post-irradiation in helasirna- <dig> cells as compared with control hela-nc cells.
depletion of dna-pkcs leads to deficiency of the transcription-coupled repair of uv-induced dna damage
on the basis of the above observation of dna damage repair in the transcribed strand of the c-myc gene in dmso-treated hl <dig> cells and dhfr gene in csb-depleted helasirna- <dig> cells, we investigated the effect of dna-pkcs on the tcr efficiency of uv radiation-damaged dna using dna-pkcs-depleted cell line hela-h <dig>  <cit> .
as shown in figure 6a, no difference in repair efficiency was observed in the untranscribed strand of the dhfr gene between dna-pkcs silenced cells  and control cells .
however, the repair efficiency of the transcribed strand was significantly decreased in hela-h <dig> cells as compared with that in control cells .
approximately 45% of the transcribed strand dna damage repair was completed at  <dig> after exposure and 80% of dna damage was repaired at  <dig> h in hela-nc cells, while the repair kinetics of dna damage in the transcribed strands was much slower in hela-h <dig> cells , suggesting a deficiency of tcr in dna-pkcs-depleted cells.
the interaction of dna-pkcs with cyclin t2
co-immunoprecipitation  using a dna-pkcs antibody was performed in hela cell extracts.
a number of dna-pkcs binding proteins were displayed by staining of the immunoprecipitates, resolved by sds-page, with coomassie bright blue .
maldi-tof-ms analysis indicated that band- <dig> was cyclin t <dig> protein , revealing the interaction of dna-pkcs with cyclin t <dig>  although the  <dig> gy γ-ray irradiated cells were used in this assay, an intensive band-5/cyclin t <dig> has also appeared in the immunoprecipitates from the untreated cells, indicating the interaction of dna-pkcs and cyclin t <dig> can occur in the normal growing condition.
therefore, we did not further perform this assay in uv-irradiation cells.
the cyclin t <dig> protein was further detected in the immunoprecipitates of dna-pkcs antibody by immunoblotting analysis using an antibody against cyclin t <dig> .
alternatively, the dna-pkcs protein has also been detected in the immunoprecipitates of cyclin t <dig> antibody by immunoblotting analysis using an antibody against dna-pkcs .
discussion
the nucleotide excision repair  pathway operates through two sub-pathways: global genome repair  and transcription-coupled repair  or gene- and strand-specific dna repair  <cit> .
the ggner is a repair mechanism which has the ability to repair dna damage to the overall genome with equivalent efficiency.
in contrast, tcr is a kind of heterogeneous dna repair, where repair to the damaged dna in the status of transcription activity is superior to the silenced genes and the repair of the transcribed strand is superior to the untranscribed strand.
some dna repair proteins and transcription factors have been identified to be involved in tcr such as csa, csb, xpg, xab <dig>  rna polymerase ii, and tfiih  <cit> .
blockage of rna polymerase □ at the dna damage site is believed to create a conducive environment for dna repair  <cit> .
in this report, we provide evidence to demonstrate that dna-pkcs, a known critical component in the nhej pathway of dna double-strand breaks, is also necessary for the tcr pathway of uv radiation-induced dna damage.
we firstly demonstrated that deficient of dna-pkcs or csb does not affect the efficiency of the global genome nucleotide excision repair .
in addition, the ggner repair kinetics of uv radiation-induced cpds in hela cells  was shown faster than that in hl <dig> cells .
a previous report indicated that the level of uvb-induced γh2ax foci  in the nonreplicating g <dig>  g <dig> and m was much higher in hela than in hl <dig> cells <cit> .
it is well known that the cyclobutane pyrimidine dimers  and pyrimidine  pyrimidone photoproducts  are the predominant lesions caused by uv radiation.
the formation of dsb in the uv-irradiated cells has been explained as resulting from  <cit> : 1) the attempted replication of dna  at the sites containing the stalling replication forks with these sites attracted an endonuclease which subsequently cleaved dna generating dsb; 2) the processing of the nucleotide excision repair  of cpds, during which the endonucleases, e.g.
ercc1-xpf, incise the adjacent cpds in inter-strand of dna to a dsb.
therefore, the formation of dsb, especially in g <dig>  g <dig> and m phases, can be considered as the intermediate product of ner, and the increased level of the transient γh2ax foci may reflect a more active ner in hela cells than in hl <dig> cells.
at present, we could not give a precise mechanistic explanation on this different kinetics of cpds repair between hela and hl <dig> cells.
in order to determine the tcr of uv-induced cyclobutane pyrimidine dimers , we firstly established a novel method of assaying tcr based on the technology of strand-specific polymerase chain reaction.
the classical method of the gene- and strand-specific dna repair or tcr assay has a tedious procedure including: dna is firstly cut with appropriate restriction enzymes, and then cut at uv-damaged cpds with t4-endonuclease v; the digested dna is electrophoresed in the alkaline agarose gels, blotted to nylon membranes; northern hybridization is performed with radioactively labeled strand-specific dna probes.
obviously, the time consuming and radioactive operation largely limit its application.
up to now, there is still no an ideal alternative method for this purpose.
this novel method has solved the major technical obstacles of time consuming and radioactive operation.
in principal, it partially evolved from the idea of the quantitative pcr detection of transcription-coupled repair based on the chromatin co-immunoprecipitated dna fragments  <cit>  and the technique of the tagged rt-pcr for the strand-specific detection of hepatitis c virus  rna  <cit> , as well as the primers design strategy of the suppression subtractive hybridization pcr method  <cit> .
this novel method only has the procedures of dna digestion with t <dig> endo v, first-strand synthesis of the single strand and the strand-specific pcr  with a set of smart specific primers as shown in table  <dig>  in order to insert an exogenous primer into the synthesized first-strand, chimera primer  was used for the first-strand synthesis of transcribed- or untranscribed-strand.
the chimera primer is composed of the upstream adaptor universal primer , which has no homology with the genomic dna of eukaryotic cells, and the downstream annealing primer .
as the tt sequence in genes easily can be damaged into tt-dimers by uv irradiation, we selected a tt-rich sequence of dhfr as a target to detect the activity of the tcr repair pathway.
after exposure to uv radiation, the genomic dna was digested with t <dig> endo v to excise the residual tt dimers, resulting in nicks in the dna strand, which led to the decrease of template dna molecules.
the first-strands of the transcribed and non-transcribed strands were synthesized separately using the specially designed primers described above.
finally, this synthesized first-strand was used as the template to amplify separately the transcribed and untranscribed strands using the primer pair of an adaptor universal primer  and a primer specifically targeting the dhfr gene.
this novel method was verified through the detection of the tcr efficiency of the dhfr gene in a csb-depleted cell line .
our data showed that depletion of csb led to a decreased efficiency of tcr to repair uv-induced dna damage in hela cells.
using our novel tcr assay, we also confirmed that the tcr efficiency of the c-myc gene was depressed when the c-myc gene transcription was silenced by dmso treatment in hl <dig> cells.
we found that deficiency of dna-pkcs does not significantly affect the efficiency of the ggner activity of uv-damaged genomic dna.
however, we observed that the tcr efficiency of the transcribed strand of the dhfr gene in the dna-pkcs-depleted hela-h <dig> cells was significantly lower than that of control hela-nc cells.
the interaction between dna-pkcs and cyclin t <dig> was identified using a co-immunoprecipitation test and a peptide fingerprint assay in this study.
cyclin t <dig> is a member of the cyclin t family, which is not directly involved in cell cycle control, but involved in the regulation of the gene transcription process through forming the human transcription elongation factor  with cdk <dig>  the cyclint1/ <dig> and cdk <dig> complex  can phosphorylate the serine  <dig> of the carboxyl-terminal domain  of rna polymerase ii and activate this enzyme, and promote transcription elongation  <cit> .
it is noteworthy that ku <dig>  another component of the dna-pk complex, can also bind to the rna polymerase □ elongation complex   <cit> .
although the authors demonstrated that the binding of ku <dig> with pol □ was not dependant on dna-pkcs  <cit> , it was evident that there is a close relationship between dna-pk and the gene transcription complex.
therefore, interaction of dna-pkcs with cyclin t <dig>  suggests that dna-pk is closely associated with the transcriptional "machinery" both physically and physiologically.
the binding of these two molecules might have two biological functions: regulation of both gene transcription and the transcription-coupled repair  mechanism.
since rna pol ii has already been verified to play an important role in the initialization of tcr  <cit> , we postulate that the involvement of dna-pkcs in tcr might be mediated through its interaction with cyclin t2/cdk9/rna pol ii.
CONCLUSIONS
a new method of tcr assay based on the strand-specific-pcr  was developed.
this new method has been validated through confirming the decreased tcr efficiency of silenced c-myc gene in dmso-treated hl <dig> cells and the dhfr gene in csb-depleted hela cells after uv irradiation.
our data suggest that dna-pkcs plays a role in the tcr pathway of uv-damaged dna.
the interaction of dna-pkcs and cyclin t <dig> was revealed.
therefore, we assume that dna-pkcs possibly functions via associating with the p-tefb complex  to modulate the activity of rna pol ii, which has already been identified as a key molecule recognizing and initializing tcr.

 cellular genomic dna constantly suffers from damage induced by various external genotoxic agents and endogenous metabolic materials. in eukaryotic cells there are multiple conserved dna repair pathways including nucleotide excision repair, which is a dna repair mechanism removing a variety of helix-distorting dna lesions including ultraviolet radiation -induced cyclobutane pyrimidine dimers, 6- pyrimidine pyrimidone photoproducts, and cigarette smoke-induced benzopyrene dna adducts. the relevance of this repair pathway is indicated by the observation that defected ner genes can result in rare human autosomal recessive disorders such as xeroderma pigmentosum and cockayne syndrome. there are two ner sub-pathways: global genomic ner repair and transcription-coupled repair, which differ mainly in the step of recognition of the dna lesions. tcr preferentially repairs the transcribed strand or transcribed genes compared to the untranscribed strand or silenced genes. in other words, the transcribed strand or genes that are undergoing transcription exhibit a faster rate of repairing dna damage than the untranscribed strand and the overall genome. rna polymerase ii plays a critical role in the recognition of dna damage in the tcr pathway. the current tcr model proposes that rna polymerase, stalled at a lesion point, directs the recruitment of repair enzymes to the transcribed strand of an active gene. this model assumes that rna polymerase ii must be removed from the lesion site of the transcribed strand to provide access for the repair complex, which initiates the repair process through unwinding the double helix at the damaged site, removal of the dna terminus, and finally filling the gap and joining the dna strands. previous studies have shown that tcr is a critical survival pathway protecting against acute toxic and long-term effects of genotoxic exposures. a number of human genetic syndromes such as xeroderma pigmentosum complementing group d and cockayne syndromes a and b have been identified as associated with a deficient tcr mechanism against uv radiation, and therefore the cells from these patients are sensitive to uv irradiation. in recent years, extensive knowledge regarding tcr has been acquired. however, as mentioned by the tcr research pioneers, hanawalt and spivak, the more we have learned, the more questions have been raised about the intricate details of tcr and its relevance to human disease. dna dependent protein kinase catalytic subunit is a member of the phosphatidylinositol 3-kinase-related protein kinase family with serine/threonine kinase activity, and it is well known as a critical component of the non-homologous end joining pathway of dna double-strand break repair. when dsb occurs in the cells, the ku80/ku heterodimer firstly recognizes and binds to the broken dna, followed by recruitment of dna-pkcs to the damage site to form the dna-pk complex with ku80/ku which consequently triggers the damage signal and subsequent dna repair. it has previously been reported that cells with mutated or decreased expression of dna-pkcs are sensitive to uv irradiation. furthermore, mouse scid cells or hamster v- cells with mutated dna-pkcs exhibit a decreased capability of ner activity, with reduced unscheduled dna synthesis after uv irradiation. however, this ner deficiency cannot be complemented by addition of dna-pk, suggesting a non-direct role of dna-pk in the ner repair steps. since rna polymerase ii is a substrate of dna-pkcs, it was suggested that dna-pk could act as a dna damage-sensor that might regulate dna repair via transcriptional modulation. in this study, we postulated that dna-pkcs might play a role in the transcription-coupled repair pathway of uv-induced cpds damage. in order to detect the tcr of ultraviolet radiation-induced dna damage, we established a novel method of tcr assay based on dna strand-specific pcr of the dihydrofolate reductase gene. we verified the efficiency and feasibility of this tcr assay in a sirna-mediated csb-depleted cell model, in which csb depletion resulted in a deficient tcr pathway. in addition, tcr of the uv damaged c-myc gene was also found to be attenuated when the c-myc gene was silenced by the treatment of dmso in hl cells. using this verified method, we demonstrated that sirna-mediated silencing of dna-pkcs has no effect on the ggner efficiency, while repair efficiency of that transcribed strand of dhfr gene was significantly lower in dna-pkcs-depleted hela-h cells than that in control hela-nc cells. furthermore, we have carried out the co-immunoprecipitation assay and identified an interaction between dna-pkcs and the cyclin t protein. cyclint together with cyclin-dependent kinase, comprises the positive transcription elongation factor b. the activated cdk phosphorylates the carboxyl terminal domain of rna pol ii, and then promotes transcription elongation. hence, we postulate that dna-pkcs may modulate the activity of rna pol ii through associating with cyclin t the present study provides new evidence suggesting the involvement of dna-pkcs in the transcription-coupled repair pathway of nucleotide excision repair in response to uv-induced dna damage.  depletion of dna-pkcs enhanced the cellular uv sensitivity as shown in the growth curve, depletion of dna-pkcs mediated by transfecting with the shrna vector expressing the specific sirna molecules targeting the catalytic motif of the dna-pkcs gene significantly decreased the growth ability of hela-h as compared to the control hela-nc cells after exposure to j/m and j/m uv irradiation. to confirm these results, a clonogenic assay was performed, and the cell survival levels also showed an increased sensitivity of the dna-pkcs-depleted hela-h cells to uv radiation as compared with the control hela-nc cells. depletion of dna-pkcs does not affect the ggner of uv-induced dna damage dna damage from uv exposure results in the frequent formation of cyclobutane pyrimidine dimers and 6- pyrimidine pyrimidone photoproducts. the thymine dimer in dna sequences can be effectively recognized and incised by t endo v. with increased repair time, the residual thymine dimers decrease, and the extent of fragmentation of genomic dna by t endo v is also decreased. therefore, detection of genomic fragmentation by the alkaline electrophoresis assay can indicate the whole genomic dna repair efficiency. as shown in figure the global genome nucleotide excision repair was completed h after uv irradiation, and there was no difference in ggner efficiency between dna-pkcs-depleted cells and control cells after j/m uv exposure. we also compared the ggner efficiency in hela cells possessing a decreased expression of the csb gene. as shown in figure 2b, depression of the csb expression mediated by sirna also had no obvious effect on ggner in helasirna- cells as compared to the control hela-nc cells. the hl cell line is sensitive to uv radiation, and the alkaline electrophoresis result confirmed that the ggner efficiency of uv-induced dna damage in hl cells is slower than in hela cells. although the dna damage was almost completely repaired at h after j/m uv radiation in hela cells, that was not accomplished in hl cells until h after exposure. development and validation of the novel method of tcr assay based on dna strand-specific pcr although depletion of dna-pkcs sensitizes hela cells to uv radiation, our results show no obvious effect on the efficiency of the ggner activity. next, we decided to focus on the transcription-coupled repair of uv-induced dna damage. the available classical method of tcr assay is time-consuming, requires running sequencing gels, and uses radioactive isotopic probes in southern blotting hybridization. here we designed a novel tcr assay based on strand-specific polymerase chain reaction with a set of smartly designed primers. this method partially evolved from the idea of the quantitative pcr detection of transcription-coupled repair based on the chromatin co-immunoprecipitated dna fragments, and the primers designing strategy of the suppression subtractive hybridization pcr method. the general theoretical basis and the technical principles are as follows: 1) it is based on the dna strand-specific pcr method to amplify the transcribed strand or untranscribed strand in the separate reaction tube with specifically designed primers for each; 2) the repair efficiency of the transcribed and untranscribed strands is reflected by the pcr efficiency of the synthesized first-strand of the transcribed or untranscribed strand using t endo v-digested genomic dna as a template, which is directly related to the repair efficiency of cpds located in dna strands. the underline sequence is an adaptor universal primer without homology to mammalian genomic dna. the experimental steps of this method are outlined in figure 3a: 1) digest the genomic dna extracted from uv exposed cells with t endo v to nick the dna at the residual cpds damage sites; 2) after dna denaturation, the first-strand dna is synthesized using the specific first-strand synthesis primer, which is composed of an upstream adaptor universal primer and a downstream annealing primer specifically targeting the active transcribed-strand or the untranscribed-strand; 3) dna elution is done to remove the free first-strand primer, and pcr amplification is carried out using the primer pairs of the up and the strand-specific down-stream primer targeting the first-strand template of the transcribed strand or the untranscribed strand; 4) analysis of the pcr products by electrophoresis. the lower efficiency of repairing the damaged dna strand, the more cpds damage sites remain. consequently, more residual damage sites provide more nicking sites for t endo v, which eventually lead to a less amount of intact dna templates for the first-strand synthesis and the pcr amplification. if both strands of the double-strand dna molecule is intact, the transcribed-strand is amplified logarithmically in the transcribed-strand pcr reaction tube due to the smart pcr primer pair of tsp and up which is appended into the templates during the first-strand synthesis of the transcribed-strand. although tsp can also anneal with the complementary untranscribed-strand, it is amplified linearly due to only one primer available for this untranscribed-strand during pcr reaction. in the same way, the untranscribed-strand is amplified logarithmically and the transcribed-strand is amplified linearly in the untranscribed-strand pcr reaction tube due to the smart pcr primer pair of ntsp and up. therefore, the transcribed strand or untranscribed strand can be easily determined in this method. concurrently, a β-actin gene fragment without a tt dimer sequence was used as an internal template reference. in order to validate the reliability and efficiency of this new method, we firstly detected the tcr efficiency of the c-myc gene in uv-irradiated hl- cells under its different transcriptional status. both mrna and protein levels of c-myc are overexpressed in hl cells. when hl cells are induced to differentiate by dmso, the transcriptional activity of c-myc is rapidly silenced. as shown in figure 4a, c-myc expression in hl cells is remarkably silenced after the treatment of dmso at ~1% and c-myc expression is almost completely inhibited after 1% dmso treatment for 48- h. for the following experiments, hl cells were treated with 1% dmso for h, and then irradiated with j/m uv. the tcr efficiency of c-myc was then detected using this new method. the results showed that the repair efficiency of the uv-damaged c-myc transcribed strand in dmso treated cells was significantly decreased as compared with the control cells without dmso treatment. this result indicates that the new tcr assay method based on strand-specific pcr is reliable for the detection of tcr of dna damage. we then used this new method to further assess the tcr of uv-induced dna damage in a cockayne syndrome b gene -depleted cell line, which was generated from hela cells by transfecting specific sirna construct to deplete the csb mrna and protein expression. csb previously has been shown to play an important role in the tcr pathway. after j/m uv radiation, strand-specific pcr of endo v enzyme digested transcribed and untranscribed strands of dhfr gene was performed. as shown in figure 5c, no difference in pcr efficiency of the untranscribed strand was observed between helasirna- and the control cells, but the pcr efficiency of the transcribed strand in helasirna- cells was obviously lower compared to that in the control cells at all repair time points post uv-irradiation. it seems that the repair was not accomplished even at h post-irradiation in helasirna- cells as compared with control hela-nc cells. depletion of dna-pkcs leads to deficiency of the transcription-coupled repair of uv-induced dna damage on the basis of the above observation of dna damage repair in the transcribed strand of the c-myc gene in dmso-treated hl cells and dhfr gene in csb-depleted helasirna- cells, we investigated the effect of dna-pkcs on the tcr efficiency of uv radiation-damaged dna using dna-pkcs-depleted cell line hela-h. as shown in figure 6a, no difference in repair efficiency was observed in the untranscribed strand of the dhfr gene between dna-pkcs silenced cells and control cells. however, the repair efficiency of the transcribed strand was significantly decreased in hela-h cells as compared with that in control cells. approximately 45% of the transcribed strand dna damage repair was completed at after exposure and 80% of dna damage was repaired at h in hela-nc cells, while the repair kinetics of dna damage in the transcribed strands was much slower in hela-h cells, suggesting a deficiency of tcr in dna-pkcs-depleted cells. the interaction of dna-pkcs with cyclin t2 co-immunoprecipitation using a dna-pkcs antibody was performed in hela cell extracts. a number of dna-pkcs binding proteins were displayed by staining of the immunoprecipitates, resolved by sds-page, with coomassie bright blue. maldi-tof-ms analysis indicated that band- was cyclin t protein, revealing the interaction of dna-pkcs with cyclin t although the gy γ-ray irradiated cells were used in this assay, an intensive band-5/cyclin t has also appeared in the immunoprecipitates from the untreated cells, indicating the interaction of dna-pkcs and cyclin t can occur in the normal growing condition. therefore, we did not further perform this assay in uv-irradiation cells. the cyclin t protein was further detected in the immunoprecipitates of dna-pkcs antibody by immunoblotting analysis using an antibody against cyclin t. alternatively, the dna-pkcs protein has also been detected in the immunoprecipitates of cyclin t antibody by immunoblotting analysis using an antibody against dna-pkcs. discussion the nucleotide excision repair pathway operates through two sub-pathways: global genome repair and transcription-coupled repair or gene- and strand-specific dna repair. the ggner is a repair mechanism which has the ability to repair dna damage to the overall genome with equivalent efficiency. in contrast, tcr is a kind of heterogeneous dna repair, where repair to the damaged dna in the status of transcription activity is superior to the silenced genes and the repair of the transcribed strand is superior to the untranscribed strand. some dna repair proteins and transcription factors have been identified to be involved in tcr such as csa, csb, xpg, xab rna polymerase ii, and tfiih. blockage of rna polymerase □ at the dna damage site is believed to create a conducive environment for dna repair. in this report, we provide evidence to demonstrate that dna-pkcs, a known critical component in the nhej pathway of dna double-strand breaks, is also necessary for the tcr pathway of uv radiation-induced dna damage. we firstly demonstrated that deficient of dna-pkcs or csb does not affect the efficiency of the global genome nucleotide excision repair. in addition, the ggner repair kinetics of uv radiation-induced cpds in hela cells was shown faster than that in hl cells. a previous report indicated that the level of uvb-induced γh2ax foci in the nonreplicating g g and m was much higher in hela than in hl cells. it is well known that the cyclobutane pyrimidine dimers and pyrimidine pyrimidone photoproducts are the predominant lesions caused by uv radiation. the formation of dsb in the uv-irradiated cells has been explained as resulting from: 1) the attempted replication of dna at the sites containing the stalling replication forks with these sites attracted an endonuclease which subsequently cleaved dna generating dsb; 2) the processing of the nucleotide excision repair of cpds, during which the endonucleases, e.g. ercc1-xpf, incise the adjacent cpds in inter-strand of dna to a dsb. therefore, the formation of dsb, especially in g g and m phases, can be considered as the intermediate product of ner, and the increased level of the transient γh2ax foci may reflect a more active ner in hela cells than in hl cells. at present, we could not give a precise mechanistic explanation on this different kinetics of cpds repair between hela and hl cells. in order to determine the tcr of uv-induced cyclobutane pyrimidine dimers, we firstly established a novel method of assaying tcr based on the technology of strand-specific polymerase chain reaction. the classical method of the gene- and strand-specific dna repair or tcr assay has a tedious procedure including: dna is firstly cut with appropriate restriction enzymes, and then cut at uv-damaged cpds with t4-endonuclease v; the digested dna is electrophoresed in the alkaline agarose gels, blotted to nylon membranes; northern hybridization is performed with radioactively labeled strand-specific dna probes. obviously, the time consuming and radioactive operation largely limit its application. up to now, there is still no an ideal alternative method for this purpose. this novel method has solved the major technical obstacles of time consuming and radioactive operation. in principal, it partially evolved from the idea of the quantitative pcr detection of transcription-coupled repair based on the chromatin co-immunoprecipitated dna fragments and the technique of the tagged rt-pcr for the strand-specific detection of hepatitis c virus rna, as well as the primers design strategy of the suppression subtractive hybridization pcr method. this novel method only has the procedures of dna digestion with t endo v, first-strand synthesis of the single strand and the strand-specific pcr with a set of smart specific primers as shown in table in order to insert an exogenous primer into the synthesized first-strand, chimera primer was used for the first-strand synthesis of transcribed- or untranscribed-strand. the chimera primer is composed of the upstream adaptor universal primer, which has no homology with the genomic dna of eukaryotic cells, and the downstream annealing primer. as the tt sequence in genes easily can be damaged into tt-dimers by uv irradiation, we selected a tt-rich sequence of dhfr as a target to detect the activity of the tcr repair pathway. after exposure to uv radiation, the genomic dna was digested with t endo v to excise the residual tt dimers, resulting in nicks in the dna strand, which led to the decrease of template dna molecules. the first-strands of the transcribed and non-transcribed strands were synthesized separately using the specially designed primers described above. finally, this synthesized first-strand was used as the template to amplify separately the transcribed and untranscribed strands using the primer pair of an adaptor universal primer and a primer specifically targeting the dhfr gene. this novel method was verified through the detection of the tcr efficiency of the dhfr gene in a csb-depleted cell line. our data showed that depletion of csb led to a decreased efficiency of tcr to repair uv-induced dna damage in hela cells. using our novel tcr assay, we also confirmed that the tcr efficiency of the c-myc gene was depressed when the c-myc gene transcription was silenced by dmso treatment in hl cells. we found that deficiency of dna-pkcs does not significantly affect the efficiency of the ggner activity of uv-damaged genomic dna. however, we observed that the tcr efficiency of the transcribed strand of the dhfr gene in the dna-pkcs-depleted hela-h cells was significantly lower than that of control hela-nc cells. the interaction between dna-pkcs and cyclin t was identified using a co-immunoprecipitation test and a peptide fingerprint assay in this study. cyclin t is a member of the cyclin t family, which is not directly involved in cell cycle control, but involved in the regulation of the gene transcription process through forming the human transcription elongation factor with cdk the cyclint1/ and cdk complex can phosphorylate the serine of the carboxyl-terminal domain of rna polymerase ii and activate this enzyme, and promote transcription elongation. it is noteworthy that ku another component of the dna-pk complex, can also bind to the rna polymerase □ elongation complex. although the authors demonstrated that the binding of ku with pol □ was not dependant on dna-pkcs, it was evident that there is a close relationship between dna-pk and the gene transcription complex. therefore, interaction of dna-pkcs with cyclin t suggests that dna-pk is closely associated with the transcriptional "machinery" both physically and physiologically. the binding of these two molecules might have two biological functions: regulation of both gene transcription and the transcription-coupled repair mechanism. since rna pol ii has already been verified to play an important role in the initialization of tcr, we postulate that the involvement of dna-pkcs in tcr might be mediated through its interaction with cyclin t2/cdk9/rna pol ii.  a new method of tcr assay based on the strand-specific-pcr was developed. this new method has been validated through confirming the decreased tcr efficiency of silenced c-myc gene in dmso-treated hl cells and the dhfr gene in csb-depleted hela cells after uv irradiation. our data suggest that dna-pkcs plays a role in the tcr pathway of uv-damaged dna. the interaction of dna-pkcs and cyclin t was revealed. therefore, we assume that dna-pkcs possibly functions via associating with the p-tefb complex to modulate the activity of rna pol ii, which has already been identified as a key molecule recognizing and initializing tcr. 
using this method, we have investigated the role of dna-dependent protein kinase catalytic subunit, a member of the phosphatidylinositol 3-kinase-related protein kinases family, in the tcr pathway of uv-induced dna damage. on the basis of the above methodology verification, we found that the depletion of dna-pkcs mediated by sirna significantly decreased the tcr capacity of repairing the uv-induced cpds damage in dhfr gene in hela cells, indicating that dna-pkcs may also be involved in the tcr pathway of dna damage repair. here we describe a new method of tcr assay based on strand-specific-pcr. to test this hypothesis, we have firstly developed a novel method of tcr assay based on the strand-specific pcr technology with a set of smart primers, which allows the strand-specific amplification of a restricted gene fragment of uv radiation-damaged genomic dna in mammalian cells. a new method of tcr assay was developed based the strand-specific-pcr. using this new method, we confirmed that sirna-mediated downregulation of cockayne syndrome b resulted in a deficiency of tcr of the uv-damaged dihydrofolate reductase gene. in eukaryotic cells, there are two sub-pathways of nucleotide excision repair, the global genome ner and the transcription-coupled repair. one possible mechanistic hypothesis is that dna-pkcs may function through associating with cyclint2/cdk to modulate the activity of rna pol ii, which has already been identified as a key molecule recognizing and initializing tcr. by means of immunoprecipitation and maldi-tof-mass spectrometric analysis, we have revealed the interaction of dna-pkcs and cyclin t which is a subunit of the human transcription elongation factor. although depletion of dna-pkcs sensitized hela cells to uv radiation, it did not affect the ggner efficiency of uv-induced cyclobutane pyrimidine dimers damage. in addition, dmso-induced silencing of the c-myc gene led to a decreased tcr efficiency of uv radiation-damaged c-myc gene in hl cells. tcr can preferentially remove the bulky dna lesions located at the transcribed strand of a transcriptional active gene more rapidly than those at the untranscribed strand or overall genomic dna. while the p-tefb complex can phosphorylate the serine of the carboxyl-terminal domain of rna polymerase ii and promote transcription elongation. our data suggest that dna-pkcs plays a role in the tcr pathway of uv-damaged dna.
 in eukaryotic cells, there are two sub-pathways of nucleotide excision repair, the global genome ner and the transcription-coupled repair. tcr can preferentially remove the bulky dna lesions located at the transcribed strand of a transcriptional active gene more rapidly than those at the untranscribed strand or overall genomic dna. this strand-specific repair in a suitable restriction fragment is usually determined by alkaline gel electrophoresis followed by southern blotting transfer and hybridization with an indirect end-labeled single-stranded probe. here we describe a new method of tcr assay based on strand-specific-pcr. using this method, we have investigated the role of dna-dependent protein kinase catalytic subunit, a member of the phosphatidylinositol 3-kinase-related protein kinases family, in the tcr pathway of uv-induced dna damage.  although depletion of dna-pkcs sensitized hela cells to uv radiation, it did not affect the ggner efficiency of uv-induced cyclobutane pyrimidine dimers damage. we postulated that dna-pkcs may involve in the tcr process. to test this hypothesis, we have firstly developed a novel method of tcr assay based on the strand-specific pcr technology with a set of smart primers, which allows the strand-specific amplification of a restricted gene fragment of uv radiation-damaged genomic dna in mammalian cells. using this new method, we confirmed that sirna-mediated downregulation of cockayne syndrome b resulted in a deficiency of tcr of the uv-damaged dihydrofolate reductase gene. in addition, dmso-induced silencing of the c-myc gene led to a decreased tcr efficiency of uv radiation-damaged c-myc gene in hl cells. on the basis of the above methodology verification, we found that the depletion of dna-pkcs mediated by sirna significantly decreased the tcr capacity of repairing the uv-induced cpds damage in dhfr gene in hela cells, indicating that dna-pkcs may also be involved in the tcr pathway of dna damage repair. by means of immunoprecipitation and maldi-tof-mass spectrometric analysis, we have revealed the interaction of dna-pkcs and cyclin t which is a subunit of the human transcription elongation factor. while the p-tefb complex can phosphorylate the serine of the carboxyl-terminal domain of rna polymerase ii and promote transcription elongation.  a new method of tcr assay was developed based the strand-specific-pcr. our data suggest that dna-pkcs plays a role in the tcr pathway of uv-damaged dna. one possible mechanistic hypothesis is that dna-pkcs may function through associating with cyclint2/cdk to modulate the activity of rna pol ii, which has already been identified as a key molecule recognizing and initializing tcr. 