title: Reduced level of ribonucleotide reductase R2 subunits increases dependence on homologous recombination repair of cisplatin-induced DNA damage.
abstract: Ribonucleotide reductase (RNR) catalyzes the rate-limiting step in the production of deoxyribonucleoside triphosphates (dNTPs) required for replicative and repair DNA synthesis. Mammalian RNR is a heteromeric enzyme consisting primarily of R1 and R2 subunits during the S phase of the cell cycle. We have shown previously that the presence of excess R2 subunits protects p53-deficient human colon cancer cells from cisplatin-induced DNA damage and replication stress. However, the mode of DNA repair influenced by changes in the level of the R2 subunit remained to be defined. In the present study, we demonstrated that depletion of BRCA1, an important factor of homologous recombination repair (HRR), preferentially sensitized stable R2-knockdown p53(-/-) HCT116 cells to the cytotoxicity of cisplatin and γ-H2AX induction. In accord with this finding, these R2-knockdown cells exhibited increased dependence on HRR, as evidenced by elevated levels of cisplatin-induced Rad51 foci and sister chromatid exchange frequency. Furthermore, stable knockdown of the R2 subunit also led to decreased cisplatin-induced gap-filling synthesis in nucleotide excision repair (NER) and a reduced dATP level in the G(2)/M phase of the cell cycle. These results suggest that an increased level of the R2 subunit extends the availability of dATP in the G(2)/M phase to promote the repair of NER-mediated single-strand gaps that are otherwise converted into double-strand breaks in the subsequent S phase. We propose that HRR becomes important for recovery from cisplatin-DNA lesions when the postexcision process of NER is restrained by reduced levels of the R2 subunit and dATP in p53-deficient cancer cells.
authors.affiliation: Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06520-8066, USA. z.ping.lin@yale.edu
authors.forename: Z Ping
authors.initials: ZP
authors.lastname: Lin
authors.forename: Yashang
authors.initials: Y
authors.lastname: Lee
authors.forename: Fang
authors.initials: F
authors.lastname: Lin
authors.forename: Michael F
authors.initials: MF
authors.lastname: Belcourt
authors.forename: Peining
authors.initials: P
authors.lastname: Li
authors.forename: Joseph G
authors.initials: JG
authors.lastname: Cory
authors.forename: Peter M
authors.initials: PM
authors.lastname: Glazer
authors.forename: Alan C
authors.initials: AC
authors.lastname: Sartorelli
mesh_terms: D000818:Animals; D002455:Cell Division / Q000235:genetics; D002945:Cisplatin / Q000633:toxicity*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D003838:Deoxyadenine Nucleotides / Q000235:genetics; D016195:G2 Phase / Q000235:genetics; D055785:Gene Knockdown Techniques; D045325:HCT116 Cells; D059765:Homologous Recombination / Q000187:drug effects / Q000235:genetics* / Q000502:physiology; D006801:Humans; D049410:Luciferases, Renilla / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency / Q000235:genetics
chemical_list: D003838:Deoxyadenine Nucleotides; D016159:Tumor Suppressor Protein p53; D049410:Luciferases, Renilla; D012264:Ribonucleotide Reductases; C108427:ribonucleotide reductase R2 subunit; C026600:2'-deoxyadenosine triphosphate; D002945:Cisplatin