title: Strand-specific PCR of UV radiation-damaged genomic DNA revealed an essential role of DNA-PKcs in the transcription-coupled repair.
abstract: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Radiation Toxicology and Oncology, Beijing Institute of Radiation Medicine, P R China.
authors.forename: Jing
authors.initials: J
authors.lastname: An
authors.forename: Tianyi
authors.initials: T
authors.lastname: Yang
authors.forename: Yuecheng
authors.initials: Y
authors.lastname: Huang
authors.forename: Feng
authors.initials: F
authors.lastname: Liu
authors.forename: Jingfen
authors.initials: J
authors.lastname: Sun
authors.forename: Yu
authors.initials: Y
authors.lastname: Wang
authors.forename: Qingzhi
authors.initials: Q
authors.lastname: Xu
authors.forename: Dechang
authors.initials: D
authors.lastname: Wu
authors.forename: Pingkun
authors.initials: P
authors.lastname: Zhou
mesh_terms: D003057:Cockayne Syndrome / Q000235:genetics; D056906:Cyclin T / Q000378:metabolism*; D042863:Cyclin-Dependent Kinase 9 / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000528:radiation effects; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase / Q000172:deficiency; D016259:Genes, myc / Q000528:radiation effects; D006367:HeLa Cells; D006801:Humans; D058539:Phosphatidylinositol 3-Kinase / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011740:Pyrimidine Dimers / Q000235:genetics / Q000528:radiation effects; D012319:RNA Polymerase II / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014158:Transcription, Genetic / Q000528:radiation effects
chemical_list: D056906:Cyclin T; D011740:Pyrimidine Dimers; D004247:DNA; D013762:Tetrahydrofolate Dehydrogenase; D058539:Phosphatidylinositol 3-Kinase; D051747:DNA-Activated Protein Kinase; D042863:Cyclin-Dependent Kinase 9; D012319:RNA Polymerase II