Long-term XPC Silencing Reduces DNA Double-Strand Break Repair

Abstract

To study the relationships between different DNA repair pathways, we established a set of clones in which one specific DNA repair gene was silenced using long-term RNA interference in HeLa cell line. We focus here on genes involved in either nucleotide excision repair (XPA and XPC) or nonhomologous end joining (NHEJ; DNA-PKcs and XRCC4). As expected, XPA KD (knock down) and XPC KD cells were highly sensitive to UVC. DNA-PKcs KD and XRCC4 KD cells presented an increased sensitivity to various inducers of double-strand breaks (DSBs) and a 70% to 80% reduction of in vitro NHEJ activity. Long-term silencing of XPC gene expression led to an increased sensitivity to etoposide, a topoisomerase II inhibitor that creates DSBs through the progression of DNA replication forks. XPC KD cells also showed intolerance toward acute ;-ray irradiation. We showed that XPC KD cells exhibited an altered spectrum of NHEJ products with decreased levels of intramolecular joined products. Moreover, in both XPC KD and DNA-PKcs KD cells, XRCC4 and ligase IV proteins were mobilized on damaged nuclear structures at lower doses of DSB inducer. In XPCproficient cells, XPC protein was released from nuclear structures after induction of DSBs. By contrast, silencing of XPA gene expression did not have any effect on sensitivity to DSB or NHEJ. Our results suggest that XPC deficiency, certainly in combination with other genetic defects, may contribute to impair DSB repair. [Cancer Res 2007;67(6):2526-34]

The Combined Effects of Xeroderma Pigmentosum C Deficiency and Mutagens on Mutation Rates in the Mouse Germ Line

“…As GRR removes a wide range of bulky DNA lesions similar to those induced by UV radiation (1, 2), some of IR-induced bulky adducts with disrupted base pairing may be substrate for this DNA-repair pathway. Besides, the results of two recent studies show that the XPC protein is also involved in the removal of oxidative DNA damage (18), the amount of which is substantially increased after irradiation, and that long-term Xpc silencing substantially reduces the efficiency of DSB repair (19). These data may provide a plausible explanation for the clinical and cellular radiosensitivity of some XPC patients (20,21).…”

Interplay between Cernunnos-XLF and Nonhomologous End-joining Proteins at DNA Ends in the Cell

“…The LIG4-defective N114P2 cells and the parental cell line Nalm-6 (gifts from Dr. M. R. Lieber, University of Southern California, Los Angeles) were isolated as described previously (15) and cultured in RPMI 1640 medium. Small interfering RNA design and cloning in pEBV-based small interfering RNA vectors carrying a hygromycin B resistance cassette and establishment of knockdown and control HeLa clones were as described elsewhere (28,29). HeLa clones were grown in Dulbecco's modified Eagle's medium in the presence of 125 g/ml hygromycin B (Invitrogen).…”

“…First, we used HeLa cells targeted for DNA-PKcs, XRCC4, or ligase IV expression and control cells as published recently (28,29). As shown in Fig.…”

A DNA-dependent stress response involving DNA-PK occurs in hypoxic cells and contributes to cellular adaptation to hypoxia

“…DNA-PKcs-deficient and DNA-PKcscomplemented cell lines (MO59-J/Fus9 and MO59-J/Fus1, respectively, a kind gift from Cordula U. Kirchgessner, Stanford University School of Medicine, Palo Alto, CA) were maintained in DMEM-F12 1:1 medium containing 15% serum. To obtain SV40 transformed MRC5 cells with long term downregulation of DNA-PKcs expression, cells were transfected with EBV-based vectors containing either control or short hairpin RNA coding sequences for human DNA-PKcs as previously described (Despras et al, 2007). The Ku80-deficient xrs-6 cell line and the xrs-6/haKu80 cell line transfected with the hamster Ku80 cDNA were obtained from the European Collection of Animal Cell Culture (Salisbury, UK).…”