DNA Translesion Synthesis Factors are Essential for Mammalian Embryonic Germ Cell Development

Germ cells are responsible for the transmission of genetic and epigenetic information between generations and hence alterations to the genome can have effects across generations. Faithful propagation of the genome is therefore of unique importance as mutations in the germline are the source of genetic variation on which evolution acts but also the drivers of sporadic, severe inherited diseases. Thus, the balance between generating genetic diversity and minimising the risk of genetic disorders is critical. However, the regulation of this balance and mechanisms for maintaining germline genome stability are incompletely understood. DNA mutations can arise from errors in replication, mis-repair of damaged DNA or the action of error-prone pathways such as DNA translesion synthesis (TLS). TLS enables DNA replication to be completed when the replication machinery stalls at impediments. In order to bypass these blocks, specialised factors are employed to ensure the completion of replication. However, due to properties of the factors involved in TLS, this occurs at the potential cost of introducing DNA mutations. The results presented in this thesis find that core components of the TLS pathway are essential in the mammalian germline but dispensable for the growth and homeostasis of somatic tissues. We find that these factors are required for fertility due to a role in primordial germ cells (PGCs). Development of PGCs entails extensive cell proliferation and in the absence of TLS initiation (Pcna^K164R/K164R or Rev1^-/-) or extension (Rev7^-/-) there is a >150-fold reduction in PGC number. Consistent with a role for TLS, PGCs in mutant embryos accumulate unresolved DNA damage and have abnormal cell cycle kinetics which are found to be unique to the germ cell compartment. Loss of PGCs in TLS-deficient embryos temporally coincides with epigenetic reprogramming. During this process, wildtype PGCs alter histone tail modifications and lose the majority of DNA methylation marks. Strikingly, we find that in the absence of TLS factors there is a failure of DNA demethylation implicating this pathway in the successful loss of methylation marks. Moreover, mutant PGCs fail to activate the germ cell transcriptional program, preventing the progression of PGC development. The findings, therefore, reveal a role for TLS factors in shaping germline genome stability and ensuring correct DNA demethylation in PGCs.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Shah, Pranay

Advisor dc:contributor.advisor

• Crossan, Gerard

• Germ cell biology

Licence

• https://www.repository.cam.ac.uk/bitstreams/e8d7279f-41d9-497d-a245-c664b2f07bb0/download
• https://www.rioxx.net/licenses/all-rights-reserved/

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.102212

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/358743