title: Breast cancer-associated missense mutants of the PALB2 WD40 domain, which directly binds RAD51C, RAD51 and BRCA2, disrupt DNA repair.
abstract: Heterozygous carriers of germ-line mutations in the BRCA2/FANCD1, PALB2/FANCN and RAD51C/FANCO DNA repair genes have an increased lifetime risk of developing 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 mutants 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 the PALB2 WD40 domain are associated with altered patterns of direct binding to the RAD51C, RAD51 and BRCA2 HR proteins in biochemical assays. Further, the T1030I missense mutant is unstable, whereas 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.
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA.
authors.forename: J-Y
authors.initials: JY
authors.lastname: Park
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Singh
authors.affiliation: 1] Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA [2] Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: N
authors.initials: N
authors.lastname: Nassar
authors.affiliation: Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA.
authors.forename: F
authors.initials: F
authors.lastname: Zhang
authors.affiliation: Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University School of Medicine, Duesseldorf, Germany.
authors.forename: M
authors.initials: M
authors.lastname: Freund
authors.affiliation: 1] Department of Otorhinolaryngology and Head/Neck Surgery, Heinrich Heine University School of Medicine, Duesseldorf, Germany [2] Unit of Pediatric Hematology/Oncology, Wells Center for Pediatric Research, Department of Pediatrics, The Riley Hospital, Indiana University School of Medicine, Indianapolis, IN, USA [3] Department of Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN, USA.
authors.forename: H
authors.initials: H
authors.lastname: Hanenberg
authors.affiliation: 1] Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA [2] Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: A R
authors.initials: AR
authors.lastname: Meetei
authors.affiliation: 1] Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Research Foundation, Cincinnati, OH, USA [2] Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
authors.forename: P R
authors.initials: PR
authors.lastname: Andreassen
mesh_terms: D024682:BRCA2 Protein / Q000737:chemistry / Q000378:metabolism*; D001943:Breast Neoplasms / Q000235:genetics*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D000076164:Fanconi Anemia Complementation Group N Protein; D005260:Female; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020125:Mutation, Missense; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D060066:Protein Interaction Maps; D051135:Rad51 Recombinase / Q000737:chemistry / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D004268:DNA-Binding Proteins; D000076164:Fanconi Anemia Complementation Group N Protein; D009687:Nuclear Proteins; C512790:PALB2 protein, human; C448305:RAD51C protein, human; D025521:Tumor Suppressor Proteins; C495153:RAD51 protein, human; D051135:Rad51 Recombinase