Analysis of cancer-associated mutations in the human POLH gene encoding  DNA polymerase η

Unrepaired DNA damage contributes to genome instability and to cancer, due to mutations in tumour suppressor genes and oncogenes. DNA damage tolerance (DDT) pathways, including translesion synthesis (TLS) carried out by the Y-family DNA polymerases Pol η, play a critical role by allowing DNA replication in the presence of template lesions, reducing replication arrest that can lead to genome instability. Inherited mutations in the POLH gene that inactivate Pol η cause xeroderma pigmentosum variant (XPV), characterised by UV-sensitivity, loss of accurate Pol η-mediated TLS, and elevated risk of skin cancer. Here, the effect of cancer-associated somatic POLH mutations on Pol η function was investigated using in silico, cell biological and biochemical approaches. 195 different somatic missense POLH mutations from the COSMIC and cBioportal databases were analysed in silico using five variant effect prediction (VEP) and four protein stability prediction programs. All mutations predicted to be deleterious by five VEP programmes were located in the N-terminal catalytic domain, except for H654N, located in the C-terminus. Seven somatic missense mutations (M14T, S51R, C72R, L89F, W174L, A223T and R361G) were deleterious in all VEP and protein stability prediction programs and may represent a subset of somatic POLH missense mutations that could affect tumour responses to DNA damaging agents. To investigate the effect of specific Pol η amino acid substitutions on the cellular response to DNA damage, a POLH-knockout MCF7 breast cancer cell line, C2, with a phenotype consistent with Polη-deficiency, was generated by CRISPR-Cas9 targeting. Transient transfection of POLH-knockout C2 cells with plasmids expressing Pol η having the somatic missense mutations, G153D, G263A, or M595V, linked to breast cancer, prostate cancer, and melanoma, respectively, showed that the mutated forms of Pol η were recruited to UV-induced nuclear foci. Using UV-induced RPA2 S4/S8 phosphorylation as marker for replication arrest-induced strand break formation, it was shown that wild-type Pol η could suppress this response, while Pol η with mutations at G263 to A, V, E, R or W did not, identifying 263 as an important somatic mutation site. Analysis of the effect of the G263A mutation on lesion bypass activity by isolated recombinant His-tagged protein Pol η showed that the G263A mutated form had 8-oxo-dG lesion bypass activity in vitro, with initial evidence of increased incorporation of dATP across from the 8-oxo-dG lesion by the G263A form. This research has identified cancer-associated somatic missense mutations in POLH that affect Pol η function in response to DNA damage. Although not tested directly here, this information on POLH mutations could be important in predicting the sensitivity of tumours to chemotherapy drugs such as cisplatin that generate damage bypassed by Pol η.

Funder

Ministry of Education, Saudi Arabia
Beckman Fund Scholarship, Ireland

Publisher

University of Galway

Rights

CC BY-NC-ND

cbn

Collections

University of Galway Theses (PhD Theses)