title: Deletion of BRCA2 exon 27 causes defects in response to both stalled and collapsed replication forks.
abstract: BRCA2 is a tumor suppressor that maintains genomic integrity through double strand break (DSB) repair and replication fork protection. The BRC motifs and an exon 27-encoded domain (Ex27) of BRCA2 interact with the recombinase RAD51 to, respectively, facilitate the formation and stability of a RAD51 filament on single strand DNA. The BRC-RAD51 associations enable DSB repair while the Ex27-RAD51 association protects the nascent replication strand from MRE11-mediated degradation. MRE11 is a nuclease that facilitates the generation of 3' overhangs needed for homologous recombination (HR)-mediated DSB repair. Here we report the dynamics of replication fork maintenance in mouse embryonic stem (ES) cells deleted for Ex27 (brca2(lex1/lex2)) after exposure to hydroxyurea (HU) that depletes nucleotides. HU conditions were varied from mild to severe. Mild conditions induce an ATR-response to replication fork stalling while severe conditions induce a DNA-PKCS-response to replication fork collapse and a DSB. These responses were differentiated by replication protein A (RPA) phosphorylation. We found that Ex27 deletion reduced MRE11 localization to stalled, but not collapsed, replication forks and that Ex27-deletion caused a proportionately more severe phenotype with HU dose. Therefore, the BRCA2 exon 27 domain maintains chromosomal integrity at both stalled and collapsed replication forks consistent with involvement in both replication fork maintenance and double strand break repair.
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, The Barshop Center of Aging, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA. Electronic address: kimt@uthscsa.edu.
authors.forename: Tae Moon
authors.initials: TM
authors.lastname: Kim
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, The Barshop Center of Aging, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
authors.forename: Mi Young
authors.initials: MY
authors.lastname: Son
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, The Barshop Center of Aging, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
authors.forename: Sherry
authors.initials: S
authors.lastname: Dodds
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, The Barshop Center of Aging, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA.
authors.forename: Lingchuan
authors.initials: L
authors.lastname: Hu
authors.affiliation: Department of Molecular Medicine, Institute of Biotechnology, The Barshop Center of Aging, The University of Texas Health Science Center at San Antonio, 15355 Lambda Drive, San Antonio, TX 78245-3207, USA. Electronic address: hastye@uthscsa.edu.
authors.forename: Paul
authors.initials: P
authors.lastname: Hasty
mesh_terms: D000818:Animals; D024682:BRCA2 Protein / Q000235:genetics*; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000378:metabolism; D005091:Exons; D017353:Gene Deletion; D042822:Genomic Instability / Q000187:drug effects / Q000235:genetics; D006918:Hydroxyurea / Q000494:pharmacology; D000076228:MRE11 Homologue Protein; D051379:Mice; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011485:Protein Binding; D051135:Rad51 Recombinase / Q000378:metabolism; D018741:Replication Origin / Q000187:drug effects / Q000235:genetics
chemical_list: D024682:BRCA2 Protein; C106711:BRCA2 protein, mouse; D004268:DNA-Binding Proteins; C512689:Mre11a protein, mouse; D019384:Nucleic Acid Synthesis Inhibitors; D051135:Rad51 Recombinase; C495154:Rad51 protein, mouse; D000076228:MRE11 Homologue Protein; D045643:DNA Repair Enzymes; D006918:Hydroxyurea
keywords: BRCA2; Chromosomal instability; Double strand break; RAD51; Replication fork stalling