Browsing by Subject "Rad51 Recombinase"

Now showing 1 - 2 of 2
  • Thumbnail Image
    Item
    BREAST CANCER-ASSOCIATED MISSENSE MUTANTS OF THE PALB2 WD40 DOMAIN, WHICH DIRECTLY BINDS RAD51C, RAD51 AND BRCA2, DISRUPT DNA REPAIR
    (Nature Publishing Group, 2014-10-02) Park, Jung-Young; Singh, Thiyam R.; Nassar, Nicolas; Zhang, Fan; Freund, Marcel; Hanenberg, Helmut; Meetei, Amom Ruhikanta; Andreassen, Paul R.; Department of Pediatrics, IU School of Medicine
    Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN, and RAD51C/FANCO DNA repair genes have an increased life-time risk to develop breast, ovarian and other cancers; bi-allelic mutations in these genes clinically manifest as Fanconi anemia (FA). Here, we demonstrate that RAD51C is part of a novel protein complex that contains PALB2 and BRCA2. Further, the PALB2 WD40 domain can directly and independently bind RAD51C and BRCA2. To understand the role of these homologous recombination (HR) proteins in DNA repair, we functionally characterize effects of missense mutations of the PALB2 WD40 domain that have been reported in breast cancer patients. In contrast to large truncations of PALB2, which display a complete loss of interaction, the L939W, T1030I, and L1143P missense mutants/variants of PALB2 WD40 domain are associated with altered direct binding patterns to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, while the L939W and L1143P proteins are stable but partially disrupt the PALB2-RAD51C-BRCA2 complex in cells. Functionally, the L939W and L1143P mutants display a decreased capacity for DNA double-strand break-induced HR and an increased cellular sensitivity to ionizing radiation. As further evidence for the functional importance of the HR complex, RAD51C mutants that are associated with cancer susceptibility and FA also display decreased complex formation with PALB2. Together, our results suggest that three different cancer susceptibility and FA proteins function in a DNA repair pathway based upon the PALB2 WD40 domain binding to RAD51C and BRCA2.
  • Thumbnail Image
    Item
    DNA Double-Strand Break Repair Genes and Oxidative Damage in Brain Metastasis of Breast Cancer
    (Oxford University Press) Woditschka, Stephan; Evans, Lynda; Duchnowska, Renata; Reed, L. Tiffany; Palmieri, Diane; Qian, Yongzhen; Badve, Sunil; Sledge, George; Gril, Brunilde; Aladjem, Mirit I.; Fu, Haiqing; Flores, Natasha M.; Gökmen-Polar, Yesim; Biernat, Wojciech; Szutowicz-Zielińska, Ewa; Mandat, Tomasz; Trojanowski, Tomasz; Och, Waldemar; Czartoryska-Arlukowicz, Bogumiła; Jassem, Jacek; Mitchell, James B.; Steeg, Patricia S.; Department of Medicine, IU School of Medicine
    Background Breast cancer frequently metastasizes to the brain, colonizing a neuro-inflammatory microenvironment. The molecular pathways facilitating this colonization remain poorly understood. Methods Expression profiling of 23 matched sets of human resected brain metastases and primary breast tumors by two-sided paired t test was performed to identify brain metastasis–specific genes. The implicated DNA repair genes BARD1 and RAD51 were modulated in human (MDA-MB-231-BR) and murine (4T1-BR) brain-tropic breast cancer cell lines by lentiviral transduction of cDNA or short hairpin RNA (shRNA) coding sequences. Their functional contribution to brain metastasis development was evaluated in mouse xenograft models (n = 10 mice per group). Results Human brain metastases overexpressed BARD1 and RAD51 compared with either matched primary tumors (1.74-fold, P < .001; 1.46-fold, P < .001, respectively) or unlinked systemic metastases (1.49-fold, P = .01; 1.44-fold, P = .008, respectively). Overexpression of either gene in MDA-MB-231-BR cells increased brain metastases by threefold to fourfold after intracardiac injections, but not lung metastases upon tail-vein injections. In 4T1-BR cells, shRNA-mediated RAD51 knockdown reduced brain metastases by 2.5-fold without affecting lung metastasis development. In vitro, BARD1- and RAD51-overexpressing cells showed reduced genomic instability but only exhibited growth and colonization phenotypes upon DNA damage induction. Reactive oxygen species were present in tumor cells and elevated in the metastatic neuro-inflammatory microenvironment and could provide an endogenous source of genotoxic stress. Tempol, a brain-permeable oxygen radical scavenger suppressed brain metastasis promotion induced by BARD1 and RAD51 overexpression. Conclusions BARD1 and RAD51 are frequently overexpressed in brain metastases from breast cancer and may constitute a mechanism to overcome reactive oxygen species–mediated genotoxic stress in the metastatic brain.