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Accumulating evidence indicates that paternal age correlates with disease risk in children. De novo gain-of-function mutations in the FGF-RAS-MAPK signaling pathway are known to cause a subset of genetic diseases associated with advanced paternal age, such as Apert syndrome, achondroplasia, Noonan syndrome, and Costello syndrome. It has been hypothesized that adult spermatogonial stem cells with pathogenic mutations are clonally expanded over time and propagate the mutations to offspring. However, no model system exists to interrogate mammalian germline stem cell competition in vivo. In this study, we created a lineage tracing system, which enabled undifferentiated spermatogonia with endogenous expression of HrasG12V, a known pathogenic gain-of-function mutation in RAS-MAPK signaling, to compete with their wild-type counterparts in the mouse testis. Over a year of fate analysis, neither HrasG12V-positive germ cells nor sperm exhibited a significant expansion compared to wild-type neighbors. Short-term stem cell capacity as measured by transplantation analysis was also comparable between wild-type and mutant groups. Furthermore, although constitutively active HRAS was detectable in the mutant cell lines, they did not exhibit a proliferative advantage or an enhanced response to agonist-evoked pERK signaling. These in vivo and in vitro results suggest that mouse spermatogonial stem cells are functionally resistant to a heterozygous HrasG12V mutation in the endogenous locus and that mechanisms could exist to prevent such harmful mutations from being expanded and transmitted to the next generation.

Maintaining genomic integrity during DNA replication is essential for cellular survival and for preventing tumorigenesis. Proliferating cell nuclear antigen (PCNA) functions as a processivity factor for DNA replication, and posttranslational modification of PCNA plays a key role in coordinating DNA repair against replication-blocking lesions by providing a platform to recruit factors required for DNA repair and cell cycle control. Here, we identify human SDE2 as a new genome surveillance factor regulated by PCNA interaction. SDE2 contains an N-terminal ubiquitin-like (UBL) fold, which is cleaved at a diglycine motif via a PCNA-interacting peptide (PIP) box and deubiquitinating enzyme activity. The cleaved SDE2 is required for negatively regulating ultraviolet damage-inducible PCNA monoubiquitination and counteracting replication stress. The cleaved SDE2 products need to be degraded by the CRL4CDT2 ubiquitin E3 ligase in a cell cycle- and DNA damage-dependent manner, and failure to degrade SDE2 impairs S phase progression and cellular survival. Collectively, this study uncovers a new role for CRL4CDT2 in protecting genomic integrity against replication stress via regulated proteolysis of PCNA-associated SDE2 and provides insights into how an integrated UBL domain within linear polypeptide sequence controls protein stability and function.