title: Conditional inactivation of the DNA damage response gene Hus1 in mouse testis reveals separable roles for components of the RAD9-RAD1-HUS1 complex in meiotic chromosome maintenance.
abstract: The RAD9-RAD1-HUS1 (9-1-1) complex is a heterotrimeric PCNA-like clamp that responds to DNA damage in somatic cells by promoting DNA repair as well as ATR-dependent DNA damage checkpoint signaling. In yeast, worms, and flies, the 9-1-1 complex is also required for meiotic checkpoint function and efficient completion of meiotic recombination; however, since Rad9, Rad1, and Hus1 are essential genes in mammals, little is known about their functions in mammalian germ cells. In this study, we assessed the meiotic functions of 9-1-1 by analyzing mice with germ cell-specific deletion of Hus1 as well as by examining the localization of RAD9 and RAD1 on meiotic chromosomes during prophase I. Hus1 loss in testicular germ cells resulted in meiotic defects, germ cell depletion, and severely compromised fertility. Hus1-deficient primary spermatocytes exhibited persistent autosomal γH2AX and RAD51 staining indicative of unrepaired meiotic DSBs, synapsis defects, an extended XY body domain often encompassing partial or whole autosomes, and an increase in structural chromosome abnormalities such as end-to-end X chromosome-autosome fusions and ruptures in the synaptonemal complex. Most of these aberrations persisted in diplotene-stage spermatocytes. Consistent with a role for the 9-1-1 complex in meiotic DSB repair, RAD9 localized to punctate, RAD51-containing foci on meiotic chromosomes in a Hus1-dependent manner. Interestingly, RAD1 had a broader distribution that only partially overlapped with RAD9, and localization of both RAD1 and the ATR activator TOPBP1 to the XY body and to unsynapsed autosomes was intact in Hus1 conditional knockouts. We conclude that mammalian HUS1 acts as a component of the canonical 9-1-1 complex during meiotic prophase I to promote DSB repair and further propose that RAD1 and TOPBP1 respond to unsynapsed chromatin through an alternative mechanism that does not require RAD9 or HUS1.
authors.affiliation: Department of Biomedical Sciences, Cornell University, Ithaca, New York, USA.
authors.forename: Amy M
authors.initials: AM
authors.lastname: Lyndaker
authors.forename: Pei Xin
authors.initials: PX
authors.lastname: Lim
authors.forename: Joanna M
authors.initials: JM
authors.lastname: Mleczko
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Diggins
authors.forename: J Kim
authors.initials: JK
authors.lastname: Holloway
authors.forename: Rebecca J
authors.initials: RJ
authors.lastname: Holmes
authors.forename: Rui
authors.initials: R
authors.lastname: Kan
authors.forename: Donald H
authors.initials: DH
authors.lastname: Schlafer
authors.forename: Raimundo
authors.initials: R
authors.lastname: Freire
authors.forename: Paula E
authors.initials: PE
authors.lastname: Cohen
authors.forename: Robert S
authors.initials: RS
authors.lastname: Weiss
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics*; D004249:DNA Damage / Q000235:genetics / Q000502:physiology; D004260:DNA Repair / Q000235:genetics; D005092:Exonucleases / Q000235:genetics / Q000378:metabolism; D005854:Germ Cells / Q000166:cytology / Q000378:metabolism; D008297:Male; D008540:Meiosis / Q000235:genetics*; D051379:Mice; D046912:Multiprotein Complexes; D013737:Testis / Q000166:cytology / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; C509939:Hus1 protein, mouse; D046912:Multiprotein Complexes; C492846:Topbp1 protein, mouse; C069930:rad9 protein; D005092:Exonucleases; C559924:Rad1 protein, mouse