Overexpression of oncogenic H-Ras in hTERT-immortalized and SV40-transformed human cells targets replicative and specialized DNA polymerases for depletion

DNA polymerases play essential functions in replication fork progression and genome maintenance. DNA lesions and drug-induced replication stress result in up-regulation and re-localization of specialized DNA polymerases eta and kappa. Although oncogene activation significantly alters DNA replication dynamics, causing replication stress and genome instability, little is known about DNA polymerase expression and regulation in response to oncogene activation. Here, we investigated the consequences of mutant H-RAS(G12V) overexpression on the regulation of DNA polymerases in h-TERT immortalized and SV40-transformed human cells. Focusing on DNA polymerases associated with the replication fork, we demonstrate that DNA polymerases are depleted in a temporal manner in response to H-RAS(G12V) overexpression. The polymerases targeted for depletion, as cells display markers of senescence, include the Pol alpha catalytic subunit (POLA1), Pol delta catalytic and p68 subunits (POLD1 and POLD3), Pol eta, and Pol kappa. Both transcriptional and post-transcriptional mechanisms mediate this response. Pol eta (POLH) depletion is sufficient to induce a senescence-like growth arrest in human foreskin fibroblast BJ5a cells, and is associated with decreased Pol alpha expression. Using an SV-40 transformed cell model, we observed cell cycle checkpoint signaling differences in cells with H-Ras(G12V)-induced polymerase depletion, as compared to Pol eta -deficient cells. Our findings contribute to our understanding of cellular events following oncogene activation and cellular transformation.

Automated summary

DNA polymerases play crucial roles in response to DNA damage and drug-induced replication stress, while oncogene activation can alter their regulation, leading to cellular senescence and affecting cell cycle checkpoint signaling.