Considerable effort has been expended in defining the molecular and cellular mechanisms governing the DNA damage response and how this pathway determines the efficacay of anti-cancer drugs. These strides have furthered drug development and someday may help physicians tailor their cancer treatment to specific diseases. The DNA damage response requires a coordinated nucleo-cytoplasmic cascade of events which ultimately converge on damaged DNA packed in chromatin. Few connections between the proteins that mediate chromatin remodeling and the proteins that mediate this damage response have been demonstrated. We have investigated the DNA damage-induced phosphorylation of KAP1, the dedicated co-repressor for all KRAB-zinc finger proteins. This proposal will bridge these two paradigms; gene silencing and DNA repair. Specifically, we have discovered that KAP1 phosphorylated at serine 824 is co-localized to numerous DNA damage response proteins at damage foci following ionizing radiation. To determine which proteins directly associate with KAP1 at these damage foci, immunoprecipitated phospho-KAP1 complexes will be analyzed using mass spectroscopy. Macromolecular complexes containing phosphorylated KAP1 will be probed for the presence of 53BP1, ZBRK1, or BRCA1 using immunoblotting. Immunofluorescence and confocal microscopy will be utilized to determine whether the s824a mutant KAP1 can form damage foci and recover the formation of 53BP1 foci following IR. Additionally, detailed studies using FACS, MTT assays, and soft agar assays will determine if the loss of KAP1 increases radiosensitivity, and if the reconstitution of KAP1 knockdown cells with s824a mutant KAP1 can restore cell viability. Lastly, this proposal will examine whether the homologous recombination DNA repair pathway is abrogated by the loss of KAP1. Relevance: Research investigating the DNA damage response continues to aid further advancements in cancer therapy. Understanding how the proteins that repair breaks in DNA are linked to the proteins that govern gene repression is important for not only for increasing our knowledge base in this critical area, but also for helping to broaden our pool of possible targets for cancer therapeutics. ? ? ?
| White, David; Rafalska-Metcalf, Ilona U; Ivanov, Alexey V et al. (2012) The ATM substrate KAP1 controls DNA repair in heterochromatin: regulation by HP1 proteins and serine 473/824 phosphorylation. Mol Cancer Res 10:401-14 |
| Ivanov, Alexey V; Peng, Hongzhuang; Yurchenko, Vyacheslav et al. (2007) PHD domain-mediated E3 ligase activity directs intramolecular sumoylation of an adjacent bromodomain required for gene silencing. Mol Cell 28:823-37 |
