In eukaryotic cells, there are two sub-pathways of nucleotide excision repair (NER), the global genome (gg) NER and the transcription-coupled repair (TCR). 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 (SS-PCR). Using this method, we have investigated the role of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a member of the phosphatidylinositol 3-kinase-related protein kinases (PIKK) 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 (CPD) 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 (DHFR) 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 HL60 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 T2, which is a subunit of the human transcription elongation factor (P-TEFb). While the P-TEFb complex can phosphorylate the serine 2 of the carboxyl-terminal domain (CTD) of RNA polymerase II and promote transcription elongation. A new method of TCR assay was developed based the strand-specific-PCR (SS-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/CDK9 (P-TEFb) to modulate the activity of RNA Pol II, which has already been identified as a key molecule recognizing and initializing TCR.
In eukaryotic cells , there are two sub - pathways of nucleotide excision repair ( NER ) , the global genome ( gg ) NER and the transcription - coupled repair ( TCR ) . 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 ( SS - PCR ) . Using this method , we have investigated the role of DNA - dependent protein kinase catalytic subunit ( DNA - PKcs ) , a member of the phosphatidylinositol 3-kinase - related protein kinases ( PIKK ) 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 ( CPD ) 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 ( DHFR ) 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 HL60 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 T2 , which is a subunit of the human transcription elongation factor ( P - TEFb ) . While the P - TEFb complex can phosphorylate the serine 2 of the carboxyl - terminal domain ( CTD ) of RNA polymerase II and promote transcription elongation . A new method of TCR assay was developed based the strand - specific - PCR ( SS - 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/CDK9 ( P - TEFb ) to modulate the activity of RNA Pol II , which has already been identified as a key molecule recognizing and initializing TCR .
Which gene strand is targeted by transcription-coupled repair (TCR)?
Which gene strand is targeted by transcription - coupled repair ( TCR ) ?
the transcribed strand
the transcribed strand