Genomic DNA is constantly damaged by exposure to exogenous and endogenous agents. Bulky adducts such as UV-induced cyclobutane pyrimidine dimers (CPDs) in the template DNA present a barrier to DNA synthesis by the major eukaryotic replicative polymerases including DNA polymerase delta. Translesion synthesis (TLS) carried out by specialized DNA polymerases is an evolutionarily conserved mechanism of DNA damage tolerance. The Y family of DNA polymerases, including DNA polymerase eta (Pol eta), the subject of this chapter, play a key role in TLS. Mutations in the human POLH gene encoding Pol eta underlie the genetic disease xeroderma pigmentosum variant (XPV), characterized by sun sensitivity, elevated incidence of skin cancer, and at the cellular level, by delayed replication and hypermutability after UV-irradiation. Pol eta is a low fidelity enzyme when copying undamaged DNA, but can carry out error-free TLS at sites of UV-induced dithymine CPDs. The active site of Pol eta has an open conformation that can accommodate CPDs, as well as cisplatin-induced intrastrand DNA crosslinks. Pol eta is recruited to sites of replication arrest in a tightly regulated process through interaction with PCNA. Pol eta-deficient cells show strong activation of downstream DNA damage responses including ATR signaling, and accumulate strand breaks as a result of replication fork collapse. Thus, Pol eta plays an important role in preventing genome instability after UV- and cisplatin-induced DNA damage. Inhibition of DNA damage tolerance pathways in tumors might also represent an approach to potentiate the effects of DNA damaging agents such as cisplatin.

Translesion synthesis by DNA polymerase eta (poleta) is one mechanism by which cancer cells can tolerate DNA damage by platinum-based anti-cancer drugs. Cells lacking poleta are sensitive to these agents. To help define the consequences of poeta-deficiency, we characterized the effects of equitoxic doses of cisplatin and carboplatin on cell cycle progression and activation of DNA damage response pathways in a human cell line lacking poleta. We show that both cisplatin and carboplatin induce strong S-phase arrest in poleta-deficient XP30RO cells, associated with reduced expression of cyclin E and cyclin B. PIK kinase-mediated phosphorylation of Chk1, H2AX and RPA2 was strongly activated by both cisplatin and carboplatin, but phosphorylation of these proteins was induced earlier by cisplatin than by an equitoxic dose of carboplatin. Compared to Chk1 and H2AX phosphorylation, RPA2 hyperphosphorylation on serine4/serine8 is a late event in response to platinum-induced DNA damage. We directly demonstrate, using dual-labeling flow cytometry, that damage-induced phosphorylation of RPA2 on serine4/serine8 occurs primarily in the S and G(2) phases of the cell cycle, and show that the timing of RPA2 phosphorylation can be modulated by inhibition of the checkpoint kinase Chk1. Furthermore, Chk1 inhibition sensitizes poleta-deficient cells to the cytotoxic effects of carboplatin. Both hyperphosphorylated RPA2 and the homologous recombination protein Rad51 are present in nuclear foci after cisplatin treatment, but these are separable events in individual cells. These results provide insight into the relationship between cell cycle regulation and processing of platinum-induced DNA damage in human cells when poleta-mediated TLS is compromised.