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.
Highlights
The requirement for effective genome maintenance is notable in germ cells, which must transmit high quality DNA to future generations
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
We propose a model in which meiotic genome maintenance involves several distinct checkpoint clamp complexes, some of which function in double-strand breaks (DSBs) repair, and others of which carry out separate functions, possibly including ATR activation through TOPBP1 in response to asynapsis
Summary
The requirement for effective genome maintenance is notable in germ cells, which must transmit high quality DNA to future generations. Meiosis, which includes the intentional generation and subsequent repair of DNA double-strand breaks (DSBs), involves a variety of DNA repair mechanisms as well as cell cycle checkpoints that monitor chromosomal integrity; much remains unknown about how these mechanisms operate in mammalian cells. We investigated how an essential DNA repair and DNA damage checkpoint complex, the RAD9-RAD1-HUS1 (9-1-1) complex, functions to maintain genome integrity in the germline. The best characterized role of 9-1-1 is in activation of ATR pathway DNA damage checkpoint signaling following replicationassociated DNA damage [2]. In response to stalled replication forks or lesions involving single-stranded DNA, the 9-1-1 complex is loaded onto 59 recessed ends by the RAD17-RFC clamp loader complex.
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