Research Article: Viral Interference with DNA Repair by Targeting of the Single-Stranded DNA Binding Protein RPA

Date Published: October 24, 2013

Publisher: Public Library of Science

Author(s): Pubali Banerjee, Rowena deJesus, Ole Gjoerup, Brian S. Schaffhausen, Michael J. Imperiale.


Correct repair of damaged DNA is critical for genomic integrity. Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. Infection with murine polyomavirus sensitizes cells to DNA damage by UV and etoposide. Polyomavirus large T antigen (LT) alone is sufficient to sensitize cells 100 fold to UV and other kinds of DNA damage. This results in activated stress responses and apoptosis. Genetic analysis shows that LT sensitizes via the binding of its origin-binding domain (OBD) to the single-stranded DNA binding protein replication protein A (RPA). Overexpression of RPA protects cells expressing OBD from damage, and knockdown of RPA mimics the LT phenotype. LT prevents recruitment of RPA to nuclear foci after DNA damage. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.

Partial Text

Because genomes are subject to different kinds of insults, cells have evolved a variety of mechanisms to repair damage [1]. Homologous recombination (HR), non-homologous end joining (NHEJ), base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR) are repair systems designed to counter different kinds of damage. Inability to correct nascent mutations is an important issue in cancer. Estimates suggest that there are from 1,000 up to 100,000 somatic mutations in common adult cancers [2].

These results point to a novel connection between DNA viruses and DNA damage regulation. LT sensitizes cells as much as one-hundred fold to DNA damage from UV irradiation or etoposide. The effect of LT is somewhat reminiscent of past reports of SV40 LT and bleomycin-induced spontaneous DNA damage [29]. LT does not modulate initial DNA damage as measured by the formation of photoproducts after UV, but rather interferes with repair. The result is excessive DNA damage revealed by the comet assays leading to apoptosis. The only unusual feature of LT induced death is the stabilization of Bim and its translocation to the nucleus. This has been seen before with Human Herpes Virus-8, which uses nuclear translocation of Bim to inhibit its activity [43]. The importance of this observation is unclear, because knockdown of Bim had no effect on phenotype. However, it remains possible that more than one member of the BH3 family is perturbed to cause the phenotype.

Antibodies against p38, phospho p38, JNK, phospho- JNK, PARP-1, BAD, BclXL, Bim, were from Cell Signaling Technology. RPA, Rad51 and Rad9 were from Santa Cruz. Anti-GFP was from Sigma. PN116 monoclonal antibody that recognizes the N-terminal domain was used to detect polyoma large T and its mutants. Anti-HA11 from Covance was used to detect HA-tagged OBD. FITC and TRITC antibody and secondary antibodies were from Jackson Immunochemicals.




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