Phosphoregulation of DSB-1 mediates control of meiotic double-strand break activity

Abstract

In the first meiotic cell division, proper segregation of chromosomes in most organisms depends on chiasmata, exchanges of continuity between homologous chromosomes that originate from the repair of programmed double-strand breaks (DSBs) catalyzed by the Spo11 endonuclease. Since DSBs can lead to irreparable damage in germ cells, while chromosomes lacking DSBs also lack chiasmata, the number of DSBs must be carefully regulated, so as to be neither too high nor too low. Here, we show that in Caenorhabditis elegans, meiotic DSB levels are controlled by the phosphoregulation of DSB-1, a homolog of the yeast Spo11 cofactor Rec114, by the opposing activities of PP4PPH-4.1 phosphatase and ATRATL-1 kinase. Increased DSB-1 phosphorylation in pph-4.1 mutants correlates with reduction in DSB formation, while prevention of DSB-1 phosphorylation drastically increases the number of meiotic DSBs both in pph-4.1 mutants as well as in the wild type background. C. elegans and its close relatives also possess a diverged paralog of DSB-1, called DSB-2, and loss of dsb-2 is known to reduce DSB formation in oocytes with increasing age. We show that the proportion of the phosphorylated, and thus inactivated, form of DSB-1 increases with age and also upon loss of DSB-2, while non-phosphorylatable DSB-1 rescues the age-dependent decrease in DSBs in dsb-2 mutants. These results suggest that DSB-2 evolved in part to compensate for the inactivation of DSB-1 through phosphorylation, to maintain levels of DSBs in older animals. Our work shows that PP4PPH-4.1, ATRATL-1, and DSB-2 act in concert with DSB-1 to promote optimal DSB levels throughout the reproductive lifespan.

Structure and DNA bridging activity of the essential Rec114–Mei4 trimer interface

“…For C. elegans , we used the two Rec114 paralogs, DSB-1 and DSB-2 (Rosu et al, 2013; Stamper et al, 2013). Consistent with an independent analysis (Guo et al, 2022), the overall folds were similar and predicted with high confidence scores: two copies of Rec114 form an approximately two-fold symmetric interlocking set of helix-turn-helix motifs embracing an a helix from Mei4. Alignment of the models for Rec114 SSM7 illustrates the strong conservation of the helix boundaries and the relative orientation between helices 2 and 3 ( Fig.…”

TOPOVIBL-REC114 interaction regulates meiotic DNA double-strand breaks

“…Although the exact REC114 effect on SPO11/TOPOVIBL organization/conformation is unknown, REC114 binding to TOPOVIBL could stabilize the SPO11/TOPOVIBL complex dimerization. This hypothesis is supported by the stoichiometry (2:1) of the Rec114/Mei4 complex in S. cerevisiae 15 and by a predicted heterotrimer from C. elegans DSB-1/ DSB-2/DSB-3 complex 42 , where DSB-1 and −2 are predicted REC114 orthologs 43,44 and DSB-3, the MEI4 ortholog 45 . Our observation that REC114 could bind simultaneously to TOPOVIBL and MEI4 (Fig.…”

“…Thus, we conclude that a potentially equivalent regulation of DSB activity could be mediated by S. cerevisiae Rec114 but with distinct molecular interactions. The regulatory function of S. cerevisiae Rec114 for DSB activity is also illustrated by the Mec1/Tel1 (ATR/ATM) dependent phosphorylation of Rec114 and its consequence on downregulating DSB activity 46 , a modification and regulation also observed for C. elegans DSB-1 42 . In A. thaliana, both the TOPOVIBL ortholog, MTOPVIB 6 , and REC114 23 have been identified.…”

Evolutionary conservation of the structure and function of meiotic Rec114−Mei4 and Mer2 complexes

“…The Rec114 PH domain, the Mei4 HEAT repeat structure, and the 2:1 Rec114–Mei4 interaction domain are generally conserved in S. pombe, M. musculus, A. thaliana , and Z. mays . In addition, AlphaFold modeling revealed a similar trimeric structure for the C. elegans homologs (DSB-1, DSB-2 and DSB-3) (Guo et al, 2022).…”