We generated knockout mice for MCM8 and MCM9 and show that deficiency for these genes impairs homologous recombination (HR)-mediated DNA repair during gametogenesis and somatic cells cycles. MCM8(-/-) mice are sterile because spermatocytes are blocked in meiotic prophase I, and females have only arrested primary follicles and frequently develop ovarian tumors. MCM9(-/-) females also are sterile as ovaries are completely devoid of oocytes. In contrast, MCM9(-/-) testes produce spermatozoa, albeit in much reduced quantity. Mcm8(-/-) and Mcm9(-/-) embryonic fibroblasts show growth defects and chromosomal damage and cannot overcome a transient inhibition of replication fork progression. In these cells, chromatin recruitment of HR factors like Rad51 and RPA is impaired and HR strongly reduced. We further demonstrate that MCM8 and MCM9 form a complex and that they coregulate their stability. Our work uncovers essential functions of MCM8 and MCM9 in HR-mediated DSB repair during gametogenesis, replication fork maintenance, and DNA repair.

Paternal and maternal epigenomes undergo marked changes after fertilization 1 . Recent epigenomic studies have revealed the unusual chromatin landscapes that are present in oocytes, sperm and early preimplantation embryos, including atypical patterns of histone modifications [2][3][4] and differences in chromosome organization and accessibility, both in gametes [5][6][7][8] and after fertilization 5,8-10 . However, these studies have led to very different conclusions: the global absence of local topological-associated domains (TADs) in gametes and their appearance in the embryo 8,9 versus the pre-existence of TADs and loops in the zygote 5,11 . The questions of whether parental structures can be inherited in the newly formed embryo and how these structures might relate to allelespecific gene regulation remain open. Here we map genomic interactions for each parental genome (including the X chromosome), using an optimized single-cell highthroughput chromosome conformation capture (HiC) protocol 12,13 , during preimplantation in the mouse. We integrate chromosome organization with allelic expression states and chromatin marks, and reveal that higher-order chromatin structure after fertilization coincides with an allele-specific enrichment of methylation of histone H3 at lysine 27. These early parental-specific domains correlate with gene repression and participate in parentally biased gene expression-including in recently described, transiently imprinted loci 14 . We also find TADs that arise in a non-parentalspecific manner during a second wave of genome assembly. These de novo domains are associated with active chromatin. Finally, we obtain insights into the relationship between TADs and gene expression by investigating structural changes to the paternal X chromosome before and during X chromosome inactivation in preimplantation female embryos 15 . We find that TADs are lost as genes become silenced on the paternal X chromosome but linger in regions that escape X chromosome inactivation. These findings demonstrate the complex dynamics of three-dimensional genome organization and gene expression during early development.We performed allele-specific single-cell HiC, modified from previous studies 12,13 , on single blastomeres (at the 1-, 2-, 4-, 8-and 64-cell stages, as well as oocytes) from highly polymorphic F 1 hybrid embryos that were obtained by crossing female Mus musculus domesticus (C57Bl/6J) with male Mus musculus castaneus CAST/EiJ) (Fig. 1 a, b). After excluding cells with poor data quality (Methods, Extended Data Fig. 1a), we used the relative coverage of the two X chromosomes to investigate sex-specific differences beyond autosomes (Extended Data Fig. 1b). Finally, we used cell cycle phasing 13 to remove cells in the pre-M and M phases, in which chromosomes lose their organization into compartments and/or domains 13,16 (Extended Data Fig. 1c-e). Looking first at the total contacts (that is, not split between alleles), we detected the formation of TAD-like domains, with clear boundaries that appear...