Higher Order Oligomerization of the Licensing ORC4 Protein Is Required for Polar Body Extrusion in Murine Meiosis

Abstract

We have previously shown that the DNA replication licensing factor ORC4 forms a cage around the chromosomes that are extruded in both polar bodies during murine oogenesis, but not around the chromosomes that are retained in the oocyte or around the sperm chromatin. We termed this structure the ORC4 cage. Here, we tested whether the formation of the ORC4 cage is necessary for polar body extrusion (PBE). We first experimentally forced oocytes to extrude sperm chromatin as a pseudo-polar body and found that under these conditions the sperm chromatin did become enclosed in an ORC4 cage. Next, we attempted to prevent the formation of the ORC4 cage by injecting peptides that contained sequences of different domains of the ORC4 protein into metaphase II oocytes just before the cage normally forms. Our rationale was that the ORC4 peptides would block protein-protein interactions required for cage formation. Two out of six tested peptides prevented the ORC4 cage formation and simultaneously inhibited polar body extrusion (PBE), resulting in the formation of two pronuclei that were retained in the oocyte. Together, these data demonstrate that ORC4 oligomerization is required to form the ORC4 cage and that it is required for PBE.

Destabilization of the replication fork protection complex disrupts meiotic chromosome segregation

“…Insults to recombination and segregation can severely compromise reproductive outcomes ( Herbert et al ., 2015 ). Increasingly, it has become apparent that factors involved in replication during cell proliferation can also regulate important aspects of the meiotic program ( Mastro and Forsburg, 2014 ; Murakami and Keeney, 2014 ; Wu and Nurse, 2014 ; Nguyen et al ., 2017 ). In this work, we observed that the FPC components are required for proper cohesion dynamics.…”

Spatial and temporal resolution of mORC4 fluorescent variants reveals structural requirements for achieving higher order self-association and pronuclei entry

“…Thus, we feel compelled to make every effort to rigorously test it. Interestingly, prevention of higher order self-association of the ORC4 complex did not prevent DNA replication suggesting it did not disrupt ORC4 DNA licensing [14]. This demonstrates that the two functions ascribed to ORC4 within embryos are not mutually exclusive and, thus, likely involve interactions within different regions of the protein.…”

“…The essential regions required for higher order self-association of ORC4 for cage formation were identified at the N-terminus and within the middle domain (figure 1; [14]). The EGFP and FlAsH mORC4 were constructed using the Gibson method (Cat No.…”

“…The function of each individual ORC protein has been primarily linked to their involvement in DNA replication, however, non-DNA licensing activity has also been noted for the ORC proteins [9][10][11][12][13]. We have reported that ORC4, a 45 kDa protein that is predicted to contain a CDC6 superfamily domain, has a role outside of DNA licensing wherein the ORC4 protein forms a cage-like structure surrounding one set of chromosomes during meiotic divisions [5,14]. DNA caged by ORC4 was observed to be expelled through polar body extrusion (PBE) and that disruption of the ORC4 self-association inhibited PBE [14].…”

“…We have reported that ORC4, a 45 kDa protein that is predicted to contain a CDC6 superfamily domain, has a role outside of DNA licensing wherein the ORC4 protein forms a cage-like structure surrounding one set of chromosomes during meiotic divisions [5,14]. DNA caged by ORC4 was observed to be expelled through polar body extrusion (PBE) and that disruption of the ORC4 self-association inhibited PBE [14]. To our knowledge, there have been no other reports of ORC4 forming a cytoplasmic cage than the two publications from our laboratory.…”