Investigating the roles of human RNaseH2 in response to oncogene- and chemotherapy-induced replication stress

Wilkins, Rosanna Jade ORCID: 0009-0003-1998-6519 (2025). Investigating the roles of human RNaseH2 in response to oncogene- and chemotherapy-induced replication stress. University of Birmingham. Ph.D.

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Abstract

RNaseH2 is a nuclear endoribonuclease that has essential roles in maintaining genome integrity in eukaryotes by degrading the RNA moiety of RNA:DNA hybrids and removing single genome-embedded ribonucleotides to prevent DNA replication stress. RNaseH2 is a heterotrimeric complex that requires all three subunits for enzymatic activity, composed of RNaseH2A, the catalytic subunit, and regulatory subunits RNaseH2B and RNaseH2C. The RNaseH2B subunit is responsible for complex nuclear localisation and interaction with PCNA. RNA:DNA hybrid levels reportedly increase in response to different replication stress-inducing agents, namely oncogenes and cancer chemotherapy drugs. There is growing interest in targeting replication stress for cancer therapy, including RNA:DNA hybrids and their binding proteins, which requires mechanistic understanding of how cancer cells tolerate increased RNA:DNA hybrids. Altered protein or mRNA expression levels of RNaseH2 subunits are implicated in multiple cancers, and their upregulation is correlated with cancer progression. However, the functions and consequences of upregulated RNaseH2 in response to replication stress remains unclear.

In this thesis, we investigate the relationship of RNaseH2 with replication stress. We demonstrate that in response to both oncogene- and chemotherapy-induced replication stress, RNaseH2 subunits are upregulated at the protein level, and correspond to increased cellular RNaseH2 activity, in human cancer and non-cancer cell lines. We generated inducible RNaseH2 subunit overexpression cell lines to model the effect of RNaseH2 upregulation and found that overexpression iii of the RNaseH2B subunit alone was the most effective strategy to increase cellular RNaseH2 activity. In response to drug-induced replication stress by camptothecin or hydroxyurea, we report that RNaseH2B overexpression can counteract drug-induced replication fork stalling and prevents additive elevation of global RNA:DNA hybrid levels. This potentially safeguards genome integrity in surviving cells in response to chemotherapy drugs, but surprisingly has limited impact on cell survival. In contrast, in response to oncogenic HRASG12V, our data suggests that upregulation of RNaseH2 is required to prevent replication fork stalling and limit cell death.

Interestingly, we discovered that RNaseH2B overexpression alone causes an unexpected increase in global RNA:DNA hybrid levels, subtle gene expression changes, mild replication stress and DNA damage in human cells, consistent with a cellular stress response.

Collectively, our data provides insight into the response of RNaseH2 subunits to replication stress, where high levels of RNaseH2 may promote tolerance to oncogene- or chemotherapy drug-induced replication stress by counteracting replication fork stalling, however, could be detrimental in unstressed cells. Therefore, increased RNaseH2 subunit proteins in cancer may be indicators of the response to chemotherapy or oncogene-induced replication stress, warranting further investigation.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):