Clamping down on mammalian meiosis

Abstract

The RAD9A-RAD1-HUS1 (9-1-1) complex is a PCNA-like heterotrimeric clamp that binds damaged DNA to promote cell cycle checkpoint signaling and DNA repair. While various 9-1-1 functions in mammalian somatic cells have been established, mounting evidence from lower eukaryotes predicts critical roles in meiotic germ cells as well. This was investigated in 2 recent studies in which the 9-1-1 complex was disrupted specifically in the mouse male germline through conditional deletion of Rad9a or Hus1. Loss of these clamp subunits led to severely impaired fertility and meiotic defects, including faulty DNA double-strand break repair. While 9-1-1 is critical for ATR kinase activation in somatic cells, these studies did not reveal major defects in ATR checkpoint pathway signaling in meiotic cells. Intriguingly, this new work identified separable roles for 9-1-1 subunits, namely RAD9A- and HUS1-independent roles for RAD1. Based on these studies and the high-level expression of the paralogous proteins RAD9B and HUS1B in testis, we propose a model in which multiple alternative 9-1-1 clamps function during mammalian meiosis to ensure genome maintenance in the germline.

Keywords: ATR; HUS1B; RAD9A-RAD1-HUS1 complex; RAD9B; TOPBP1; double-strand break repair; meiosis; pachytene checkpoint; synapsis.

Figure 1. RAD9A and RAD1 proteins localize in distinct yet overlapping patterns on mammalian meiotic chromosomes. The localization patterns of RAD9A and RAD1 are shown in schematic form (left) and in representative immunofluorescence images (right).(A)During late pachynema, unsynapsed regions of the X and Y axial elements are continuously coated with RAD1, while the X chromosome axial element additionally harbors discrete RAD9A foci, presumably at sites of DSBs.(B)On early pachytene-stage autosomes, colocalization of RAD9A and RAD1 is observed in a focal pattern, likely marking DSB sites.(C)Asynapsed regions of autosomes show extensive RAD1 staining and discrete RAD9A foci. Similar to what occurs on the unsynapsed X and Y (A), RAD9A is present at sites that also contain RAD1, whereas RAD1 displays a broader staining pattern that includes regions without detectable RAD9A. See reference for additional examples of these staining patterns.

Figure 2. Proposed functions for canonical and alternative 9-1-1 complexes during mammalian meiosis.(A)We propose that the canonical RAD9A-RAD1-HUS1 complex is loaded on at least a subset of DSB sites and facilitates DSB repair and potentially checkpoint signaling. Alternative trimeric 9-1-1 complexes, containing RAD1 and a combination of RAD9B, RAD9A, HUS1B, or HUS1, may form in mouse germ cells and localize to asynapsed chromatin to facilitate meiotic silencing through MSUC and MSCI. The composition of this complex has yet to be determined, but here we propose a model involving RAD9B-RAD1-HUS1B. Depiction is based on the crystal structure of human 9-1-1.^-(B)Based on the observed localization of RAD9A and RAD1 along asynapsed autosomes, we hypothesize that discrete RAD9A foci represent conventional 9-1-1 complexes at DSB sites, while more extensive RAD1 staining at regions of asynapsis reflects the presence of alternative clamps that include RAD1 in conjunction with paralogs HUS1B and/or RAD9B, without HUS1 or RAD9A.