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http://purl.uniprot.org/citations/21214942http://www.w3.org/1999/02/22-rdf-syntax-ns#typehttp://purl.uniprot.org/core/Journal_Citation
http://purl.uniprot.org/citations/21214942http://www.w3.org/2000/01/rdf-schema#comment"

Background

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.

Results

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.

Conclusion

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."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.org/dc/terms/identifier"doi:10.1186/1471-2091-12-2"xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"An J."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Huang Y."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Liu F."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Wu D."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Sun J."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Wang Y."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Xu Q."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Yang T."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/author"Zhou P."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/date"2011"xsd:gYear
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/name"BMC Biochem"xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/pages"2"xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/title"Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair."xsd:string
http://purl.uniprot.org/citations/21214942http://purl.uniprot.org/core/volume"12"xsd:string
http://purl.uniprot.org/citations/21214942http://www.w3.org/2004/02/skos/core#exactMatchhttp://purl.uniprot.org/pubmed/21214942
http://purl.uniprot.org/citations/21214942http://xmlns.com/foaf/0.1/primaryTopicOfhttps://pubmed.ncbi.nlm.nih.gov/21214942
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