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.

Author Summary

Meiosis is a specialized cell division process in which germ cells undergo two cell divisions to produce haploid progeny. Two processes, genetic recombination and chromosome pairing/synapsis, are critical for successful meiosis and the production of gametes with high chromosomal integrity. The RAD9-RAD1-HUS1 (9-1-1) complex has been proposed to play critical roles in recombination as well as in the checkpoint-dependent monitoring of chromosomal synapsis by facilitating activation of the ATR checkpoint kinase. Our data indicate that HUS1 is required for normal germ cell development and fertility, for efficient completion of a subset of meiotic DNA recombination events, and for proper exclusion of the non-sex chromosomes from a specialized, repressive chromatin domain containing the X and Y chromosomes. However, HUS1 is not required for the meiotic functions of ATR in responding to chromosome synapsis defects. Furthermore, RAD1 localizes to sites along asynapsed chromosomes that lack detectable RAD9, and does so in the absence of Hus1, implicating RAD1 in a novel response to unsynapsed chromatin that is independent of the canonical 9-1-1 complex. Since mice lacking Hus1 in germ cells exhibit chromosomal abnormalities and severely reduced fertility, this work has broad implications for the maintenance of genome stability in the germline and for human reproductive health.