ATR signaling in mammalian meiosis: From upstream scaffolds to downstream signaling

Abstract

In germ cells undergoing meiosis, the induction of double strand breaks (DSBs) is required for the generation of haploid gametes. Defects in the formation, detection, or recombinational repair of DSBs often result in defective chromosome segregation and aneuploidies. Central to the ability of meiotic cells to properly respond to DSBs are DNA damage response (DDR) pathways mediated by DNA damage sensor kinases. DDR signaling coordinates an extensive network of DDR effectors to induce cell cycle arrest and DNA repair, or trigger apoptosis if the damage is extensive. Despite their importance, the functions of DDR kinases and effector proteins during meiosis remain poorly understood and can often be distinct from their known mitotic roles. A key DDR kinase during meiosis is ataxia telangiectasia and Rad3-related (ATR). ATR mediates key signaling events that control DSB repair, cell cycle progression, and meiotic silencing. These meiotic functions of ATR depend on upstream scaffolds and regulators, including the 9-1-1 complex and TOPBP1, and converge on many downstream effectors such as the checkpoint kinase CHK1. Here, we review the meiotic functions of the 9-1-1/TOPBP1/ATR/CHK1 signaling pathway during mammalian meiosis.

Keywords: 9-1-1 complex; ATR; CHK1; DNA damage signaling; TOPBP1; double-stranded DNA break; homologous recombination; meiosis; synapsis.

Figure 1.. Key mitotic and meiotic functions of ATR.

The schematic highlights ATR functions throughout mitotic (A) and meiotic (B) cell cycles. A) During a mitotic cell cycle, a diploid cell replicates and then divides to form two daughter diploid cells. ATR plays a role in several key steps throughout the cell cycle, enforcing the G1/S, intra-S, and G2/M checkpoints and mediating other key functions related to DNA replication and repair. B) During a meiotic cell cycle, diploid germ cells undergo DNA replication, followed by meiosis I during which cells undergo recombination of homologous chromosomes and synapsis, ultimately yielding two daughter cells. These cells then enter meiosis II, with the subsequent division producing four haploid cells. ATR has similar functions in pre-meiotic and mitotic cells and additionally is critical during meiosis for the silencing of unsynapsed chromatin. C) Meiotic prophase I has 5 sub-stages: leptonema, zygonema, pachynema, diplonema, and diakinesis. Upon double strand DNA break (DSB) induction by SPO11 (for simplicity a single break is shown here), the pairing and recombination of homologous chromosomes is initiated. Synapsis of homologous chromosomes begins to occur in zygonema and is mediated by the synaptonemal complex (SC) lateral and central elements. At pachynema homologous chromosomes are completely synapsed and crossover (CO) recombination is completed.

Figure 2.. ATR activation during the mitotic cell cycle.

During a mitotic cell cycle ATR can be activated by either 9A-1-1/TOPBP1 or ETAA1. As an example, 5’ to 3’ resection of the DNA ends at a DSB creates ssDNA overhangs that can be coated with RPA. ATRIP then recruits ATR to the RPA-coated ssDNA. The 9A-1-1 complex is loaded at recessed 5’ ends by the RAD17-RFC clamp loader and interacts with TOPBP1, resulting in ATR activation. Alternatively, ETAA1 can be recruited to RPA-coated ssDNA and directly activate ATR. Upon activation, ATR-mediated signaling triggers downstream events critical for proper cell cycle progression and cell survival.

Figure 3:. Proposed mechanism of meiotic silencing via ATR signaling.

Synaptonemal complex (SYCP1/SYCP3) staining of a wild-type mouse spermatocyte illustrates completed autosomal synapsis indicated by the presence of SYCP1 on the chromosome cores. The X and Y chromosomes synapse at a small region called the pseudoautosomal region (PAR), but the remainder of the X and Y chromosomes remain unsynapsed and therefore lack SYCP1 staining. The unsynapsed region is coated by HORMAD1 and HORMAD2. ATR is recruited via HORMADs and BRCA1 and phosphorylates H2AX (as shown in the stained meiotic spread), which localizes along with MDC1 to the chromatin loops.

Figure 4:. Proposed mechanisms of ATR activation at meiotic DSB.

During a mitotic cell cycle, ATR activation can be mediated via 9-1-1/TOPBP1 or ETAA1 at resected DNA ends. Whether the same mechanisms are active at SPO11-induced meiotic DSB remains unknown. In mitotic cells, the 9-1-1 complex is loaded at the junction of dsDNA and ssDNA breaks by the RAD17-RFC clamp loader and is stabilized with the help of RHINO. 9-1-1/TOPBP1 interaction initiates ATR activation via the AAD of TOPBP1. The precise role of the 9-1-1 complex in ATR activation during meiosis remains unclear. Evidence suggests that the 9-1-1 complex and TOPBP1 are present at meiotic DSB and promote DSB repair. ETAA1 can also mediate ATR activation independently of the 9-1-1 complex, but the contributions of ETAA1 to ATR activation during meiosis remain largely unexplored.